Preparation of psilocybin, different polymorphic forms, intermediates, formulations and their use

ABSTRACT

This invention relates to the large-scale production of psilocybin for use in medicine. More particularly, it relates to a method of obtaining high purity crystalline psilocybin, particularly, in the form of Polymorph A. It further relates to a method for the manufacture of psilocybin and intermediates in the production thereof and formulations containing psilocybin.

This application is a continuation of U.S. patent application Ser. No.16/155,386, filed Oct. 9, 2018, which claims priority to United KingdomApplication No. 1816438.4, filed Oct. 9, 2018; United KingdomApplication No. 1810588.2, filed Jun. 28, 2018; and United KingdomApplication No. 1716505.1, filed Oct. 9, 2017. The disclosures of theabove applications are incorporated by reference herein in theirentireties.

This invention relates to the large-scale production of psilocybin foruse in medicine.

By large scale is meant producing batches of psilocybin with a weight ofgreater than 10 g, more preferably greater than 100 g, more preferablystill greater than 250 g and up to, and above, Kg levels.

It also relates to the production of intermediates, including but notlimited to psilocin, different polymorphic forms of psilocybin,including isostructural variants, and their formulation for use inmedicine, particularly, but not exclusively for the treatment oftreatment resistant depression, as defined in Diagnostic and StatisticalManual, 5^(th) Edition, either alone or in combination withpsychological support which may be provided digitally.

BACKGROUND

Psilocybin was first synthesised in 1958 by Sandoz, see GB912714 andU.S. Pat. No. 3,075,992, and was widely available as a research chemicaluntil the mid-1960's.

A plant based psychedelic it has been used as an aide to psychotherapyfor the treatment of mood disorders and alcoholic disorders and recently3 clinical trials have reported its use for depressive symptoms.

-   Griffiths et al 2016; J Psychopharmacol 30 (12):1181-1197;-   Ross et al 2016; J Psychopharmacol 30 (12):1165-1180; and-   Carhart-Harris et al 2016, Lancet Psychiatry 3(7): 619-627.-   Methods of manufacture of psilocybin are limited and include:-   J Nat Prod 2003, 66, pages 885-887;-   Helv Chim Acta 1959, 42, 2073-2103;-   Experientia 1958, 15, 397-399; and-   Synthesis 1999, 935-938.

Based on this literature Applicant believed that the process disclosedin J Nat Prod 2003, 66, pages 885-887 (hereafter JNP) was the mostsuitable method for development into a commercial scaled process.

The process disclosed therein produced quantities in the order of 10 gand comprised 6 steps numbered (i) to (vi).

By analogy with the Applicants process, steps ii and iii are hereafterdiscussed as a single step, (Step 2) and the JNP process is reproducedas FIG. 1 herein.

Step 1 (i) comprised reacting 4-hydroxyindole (“3”) with aceticanhydride (Ac₂O) in pyridine and anhydrous dichloromethane (CH₂Cl₂) atOPC. Water was added, the mixture evaporated, and the resultingconcentrate was dissolved in ethyl acetate, washed with water, andsaturated sodium chloride, and the organic phase dried over sodiumsulphate and evaporated to obtain 4-acetylindole (“4”), which wascollected by filtration and washed with water and ethyl acetate.

Step 2 (ii and iii), a two-step acylation (ii)—amidation step (iii),comprised forming 3-Dimethylaminooxalyl-4-acetylindole (“6”) by: (ii)reacting 4-acetylindole (“4”) with oxalyl chloride ((COCl)₂) inanhydrous diethylether, stirring, adding n-hexane and holding at −20° C.to produce an intermediate 3-(2-chloro-2-oxoacetyl)-1H-indol-4-ylacetate (“5”) which was separated by filtration. The intermediate wasdissolved in anhydrous tetrahydrofuran (THF) and reacted withdimethylamine ((CH₃)₂NH) in tetrahydrofuran and pyridine. Anhydrousether was added because of solidification, and the reaction productseparated by filtration and washed with n hexane, ethyl acetate, andwater to obtain 3-Dimethylaminooxalyl-4-acetylindole (“6”).

Step 3 (iv) comprised the formation of psilocin (“1”) by reacting the3-Dimethylaminooxalyl-4-acetylindole (“6”) with lithium aluminiumhydride (LiAlH₄) in anhydrous THF under an argon atmosphere. Afterrefluxing and cooling, anhydrous sodium sulphate was added, followed bya solution of sodium sulphate, and further anhydrous sodium sulphate.The reaction mixture was diluted with ethyl acetate, quicklyconcentrated in vacuo, and the resulting psilocin crystals brieflywashed with methanol.

Step 4 (v) comprised the formation of benzyl [2-(4-oxyindol-3-yl)ethyl]dimethylammonio-4-O-benzyl phosphate (“8”) by reacting psilocin,dissolved in anhydrous THF, with n-butyl lithium (n-BuLi) in n-hexane at−78° C. and tetrabenzylpyrophosphate [(BnO)₂PO]₂O, and the reactionallowed to warm to OPC, and the production of intermediate dibenzyl3-[2-(dimethylamino)ethyl]-1H-indol-4-yl phosphate (“7”) monitored. Onchecking for its presence, aminopropyl silica gel was added, the mixturediluted with ethyl acetate and filtered through a Celite pad by suction,the filtrate concentrated in vacuo, re-dissolved in CH₂Cl₂, and theprecipitate collected by filtration.

Step 5 (vi) comprised the formation of psilocybin (“2”) by reaction of(“8”), in methanol (MeOH), with hydrogen (H₂) using apalladium-activated carbon catalyst (Pd/C). Water was added, because ofproduct deposition, and (“8”), its mono de-benzylated derivative weremonitored along with the appearance of psilocybin, the reaction solutionwas filtered through a Celite pad. The product was collected byfiltration and washed with ethanol to provide a white needle crystallineform with a melting point 190° C.-198° C.

In contrast to most processes, such as JNP, which use non-aqueoussolvents, such as methanol or ethanol, Experientia 1958, 15, 397-399used a single re-crystallisation from water to obtain psilocybin from amushroom extraction. The teaching was to use boiling water to dissolvethe starting material, obtained at small scale by chromatography, andthe resulting high vacuum dried material was stated to melt indistinctlybetween 185 and 195° C., and showed a weight loss of 25.4%, suggestingit clearly differs in purity and form to that obtained by Applicant.

During the development of a synthesis to produce Psilocybin theapplicant conducted a number of hydrogenation reactions on a 5 g scalewhich resulted in different crystalline forms of Psilocybin beingobtained. The initial hydrogenation reaction yielded Hydrate A(JCCA2157E) which exhibited a XRPD diffractogram as shown in FIG. 7d andDSC and TGA thermograms as shown in FIG. 8d . The DSC exhibits anendotherm at ˜97° C. which is coincidental with a weight reduction inthe TGA indicative of dehydration, and an endothermic event with anonset temperature of ˜216° C. which was presumed to be the melt. Anotherhydrogenation reaction yielded an ethanol solvate (JCCA2158D) which whenanalysed by XRPD (FIG. 7e ), DSC (FIG. 8e ), TGA (FIG. 8e ) and by ¹HNMR indicated 11% entrapped ethanol. The DSC thermogram shows anendotherm having an onset of ˜154° C. that appeared to be a meltconcurrent with the ˜13% weight loss in the TGA. In another experimentperformed during development, the applicant performed a crystallisationof psilocybin; rather than remain in solution in hot water allowing fora polish filtration step, precipitation occurred at high temperature(>90° C.). The solids formed did not re-dissolve upon further heating oraddition of extra water. Upon cooling and isolation of the solid(CB646E) XRPD was performed. The XRPD diffractogram (FIG. 7f ) suggesteda mixed phase of Polymorph A′ (JCCA2160-F-D4) and Polymorph B(JCCA2160-F-TM2-C5). These findings highlight the importance ofdeveloping a process which can consistently produce the desiredcrystalline form so the Applicants set about experiments to determinewhat these forms were in order they could produce a chemically purepsilocybin, in a controlled form suitable for use in medicine.

For clinical trials any New Active Substance (NAS) should be capable oflarge scale production (typically 100 g plus, more typically greaterthan 250 g, more preferably still greater than 500 g, to Kg plusbatches), depending on the amount of active to be dosed to a humansubject. It should also be chemically pure, well defined, and stable onstorage.

Furthermore, any method of manufacture must be readily reproducible, andprovide batch to batch consistency.

It is a first object of this invention to provide psilocybin, ofconsistent polymorphic form, for administration to human subjects.

It is another object of this invention to provide chemically purepsilocybin, of consistent polymorphic form, for administration to humansubjects.

It is yet a further object to provide chemically pure psilocybin, inlarge scale batch quantities since for commercial use, the purepsilocybin must be produced at scale.

It is yet a further object of the invention to provide a method ofcrystallising psilocybin in a desired polymorphic form.

It is yet a further object of the present invention to provide ascalable method for manufacturing psilocybin, from psilocin or 4hydroxy-indole.

In developing suitable methodology Applicant experienced numerousproblems and difficulties which they had to be overcome, and it is aseparate, independent, object to overcome those problems identified ateach step, and use the inventions either alone or in combination.

It is yet a further object of the invention to formulate the psilocybinof the invention in a form suitable for administration to human subjectsand use it in medicine, particularly in the treatment of central nervoussystem disorders (CNS), and more particularly, but not exclusively, inthe treatment of depression, particularly, drug resistant depressioneither alone or in combination with a digital health product or digitalsolution.

BRIEF SUMMARY OF THE DISCLOSURE

In accordance with a first aspect of the present inventions there isprovided crystalline psilocybin in the form Polymorph A or Polymorph A′,characterised by one or more of:

a. peaks in an XRPD diffractogram at 11.5, 12.0 and 14.5 °2θ±0.1 °2θ;

b. peaks in an XRPD diffractogram at 11.5, 12.0 and 14.5 °2θ±0.1 °2θ,further characterised by at least one further peak at 19.7, 20.4, 22.2,24.3 or 25.7 °2θ±0.1 °2θ;

c. an XRPD diffractogram as substantially illustrated in FIG. 7a or 7 b;or

d. an endothermic event in a DSC thermogram having an onset temperatureof between 205 and 220° C. substantially as illustrated in FIG. 8a or 8b.

Polymorph A

In accordance with a preferred embodiment of the present invention thereis provided crystalline psilocybin in the form Polymorph A,characterised by one or more of:

-   -   a. peaks in an XRPD diffractogram at 11.5, 12.0, 14.5, and 17.5,        °2θ±0.1 °2θ;    -   b. peaks in an XRPD diffractogram at 11.5, 12.0, 14.5 and 17.5,        °2θ±0.1 °2, further characterised by at least one further peak        at 19.7, 20.4, 22.2, 24.3 or 25.7 °2θ±0.1 °2θ;    -   c. an XRPD diffractogram as substantially illustrated in FIG. 7a        ; or    -   d. an endothermic event in a DSC thermogram having an onset        temperature of between 205 and 220° C. substantially as        illustrated in FIG. 8 a.

The peak at 17.5 °2θ±0.1 °2θ has a relative intensity compared to thepeak at 14.5 °2θ±0.1 °2θ of at least 5%, preferably at least 6%, morepreferably still at least 7%, through 8%, and 9% to at least 10%.

In one embodiment, psilocybin Polymorph A exhibits an XRPD diffractogramcharacterised by the diffractogram summarised in Table 1. In oneembodiment, described herein, the crystalline psilocybin Polymorph Acomprises at least 3 peaks of (±0.1 °2θ) of Table 1. In a certainembodiment, described herein, the crystalline psilocybin Polymorph Acomprises at least 4 peaks of (±0.1 °2θ) of Table 1. In a certainembodiment, described herein the crystalline psilocybin Polymorph Acomprises at least 5 peaks of (±0.1 °2θ) of Table 1. In a certainembodiment, described herein the crystalline psilocybin Polymorph Acomprises at least 6 peaks of (±0.1 °2θ) of Table 1. In a certainembodiment, described herein the crystalline psilocybin Polymorph Acomprises at least 8 peaks of (±0.1 °2θ) of Table 1. In a certainembodiment, described herein the crystalline psilocybin Polymorph Acomprises at least 10 peaks of (±0.1 °2θ) of Table 1. In a certainembodiment, described herein the crystalline psilocybin Polymorph Acomprising at least 15 peaks of (±0.1 °2θ) of Table 1. A peak at about17.5, °2θ±0.1 °2θ distinguishes psilocybin Polymorph A from PolymorphA′, in which the peak is absent or substantially absent (i.e. has arelative intensity compared to the peak at 14.5 °2θ±0.1 °2θ of less than2%, more preferably less than 1%).

TABLE 1 XRPD peak positions for Polymorph A Relative Intensity Position[°2 Th.] [%] 5.6 8.42 11.5 13.05 12.0 26.45 14.5 100 17.5 10.71 19.737.29 20.4 20.06 22.2 17.83 23.2 6.99 24.3 17.93 25.7 16.4 26.8 3.1527.8 4.54 29.7 9.53 31.2 6.51 32.6 2.45 33.7 1.75

In one embodiment, crystalline psilocybin Polymorph A is characterisedby XRPD diffractogram peaks at 11.5, 12.0, 14.5, and 17.5 °2θ±0.1 °2θ.In another embodiment, crystalline psilocybin Polymorph A is furthercharacterised by at least one additional peak appearing at 19.7, 20.4,22.2, 24.3 or 25.7 °2θ±0.1 °2θ. In another embodiment, crystallinepsilocybin Polymorph A is further characterised by at least twoadditional peaks appearing at 19.7, 20.4, 22.2, 24.3 or 25.7 °2θ±0.1°2θ. In another embodiment, crystalline psilocybin Polymorph A isfurther characterised by at least three additional peaks appearing at19.7, 20.4, 22.2, 24.3 or 25.7 °2θ±0.1 °2θ. In yet a further embodiment,crystalline psilocybin Polymorph A exhibits an XRPD diffractogramsubstantially the same as the XRPD diffractogram shown in FIG. 7 a.

In one embodiment, crystalline psilocybin Polymorph A is characterisedby XRPD diffractogram peaks at 14.5 and 17.5 °2θ±0.1 °2θ with the peakat 17.5 °2θ having an intensity which is at least 5% of the intensity ofthe peak at 14.5 °2θ, more preferably still at least 6%, through atleast 7%, at least 8%, at least 9%, to at least 10%.

In one embodiment, crystalline psilocybin Polymorph A is absent orsubstantially absent of an XRPD diffractogram peaks at 10.1. Bysubstantially absent is meant than any XRPD diffractogram peaks at 10.1is less than 2% of the intensity of the peak at 14.5 °2θ, such as lessthan 1%, or is not detectable in the XRPD diffractogram,

In one embodiment, crystalline psilocybin Polymorph A is characterisedby an endothermic event in a DSC thermogram having an onset temperatureof between 205 and 220° C., such as between 210 and 220° C., such asbetween 210 and 218° C., or such as between 210 and 216° C. In anotherembodiment, crystalline psilocybin Polymorph A is further characterisedby an endothermic event in the DSC thermogram having an onsettemperature of between 145 and 165° C., such as between 145 and 160° C.,or such as between 145 and 155° C. In another embodiment, crystallinepsilocybin Polymorph A is characterised by an endothermic event havingan onset temperature of between 205 and 220° C., such as between 210 and220° C., such as between 210 and 218° C., or such as between 210 and216° C., and an endothermic event having an onset temperature of between145 and 165° C., such as between 145 and 160° C., or such as between 145and 155° C., in a DSC thermogram. In yet another embodiment, crystallinepsilocybin Polymorph A exhibits a DSC thermogram substantially the sameas the DSC thermogram in FIG. 8 a.

In another embodiment, crystalline psilocybin Polymorph A ischaracterised by having a water content of <0.5% w/w, such as <0.4% w/w,such as <0.3% w/w, such as <0.2% w/w, or such as <0.1% w/w. The skilledperson would know of methods to determine the water content of acompound, for example Karl Fischer Titration. In one embodiment,crystalline psilocybin Polymorph A is characterised by having <0.5% w/wloss, such as <0.4% w/w, such as <0.3% w/w, such as <0.2% w/w, such as<0.1% w/w, in the TGA thermogram between ambient temperature, such asabout 25° C., and 200° C. In one embodiment, crystalline psilocybinPolymorph A loses less than 2% by weight in a loss on drying test, suchas less than 1% by weight, such as less than 0.5% by weight. The loss ondrying test is performed at 70° C.

In one embodiment, crystalline psilocybin Polymorph A is a highly purecrystalline form of Polymorph A, for example, psilocybin comprises atleast 90% by weight, such as 95%, such as 99%, such as 99.5% ofPolymorph A.

In one embodiment, crystalline psilocybin Polymorph A is a white to offwhite solid.

In another embodiment, crystalline psilocybin Polymorph A is chemicallypure, for example the psilocybin has a chemical purity of greater than97%, such as greater than 98%, or such as greater than 99% by HPLC. Inone embodiment, crystalline psilocybin Polymorph A has no singleimpurity of greater than 1%, more preferably less than 0.5%, includingphosphoric acid as measured by ³¹P NMR, and psilocin as measured byHPLC. In one embodiment, crystalline psilocybin Polymorph A has achemical purity of greater than 97 area %, more preferably still greaterthan 98 area %, and most preferably greater than 99 area % by HPLC. Inone embodiment, crystalline psilocybin Polymorph A has no singleimpurity greater than 1 area %, more preferably less than 0.5 area % asmeasured by HPLC. In one embodiment, crystalline psilocybin Polymorph Adoes not contain psilocin at a level greater than 1 area %, morepreferably less than 0.5 area % as measured by HPLC. In one embodiment,crystalline psilocybin Polymorph A does not contain phosphoric acid at alevel greater than 1 weight %, more preferably less than 0.5 weight % asmeasured by ³¹P NMR. In one embodiment, crystalline psilocybin PolymorphA has a chemical assay of at least 95 weight %, such as at least 96weight %, or such as at least 98 weight %.

Polymorph A′

In accordance with another embodiment of the invention, there isprovided crystalline psilocybin Polymorph A′ characterised by one ormore of:

-   -   a. peaks in an XRPD diffractogram at 11.5, 12.0 and 14.5 °2θ±0.1        °2θ, but absent or substantially absent of a peak at 17.5        °2θ±0.1 °2θ;    -   b. peaks in an XRPD diffractogram at 11.5, 12.0 and 14.5 °2θ±0.1        °2θ, but absent or substantially absent of a peak at 17.5        °2θ±0.1 °2θ, further characterised by at least one further peak        at 19.7, 20.4, 22.2, 24.3 or 25.7 °2θ±0.1 °2θ;    -   c. an XRPD diffractogram as substantially illustrated in FIG. 7b        ; or    -   d. an endothermic event in a DSC thermogram having an onset        temperature of between 205 and 220° C. substantially as        illustrated in FIG. 8 b.

By substantially absent of a peak at 17.5 °2θ±0.1 °2θ is meant, ifpresent, the peak has a relative intensity, compared to a peak at 14.5°2θ±0.1 °2θ, of less than 5%, more preferably less than 4%, through lessthan 3%, to 2%, 1% or less.

In one embodiment, psilocybin Polymorph A′ exhibits an XRPDdiffractogram characterised by the diffractogram summarised in Table 2.In one embodiment, described herein the crystalline psilocybin PolymorphA′ comprises at least 3 peaks of (±0.1 °2θ) of Table 2 but absent orsubstantially absent of a peak at 17.5 °2θ±0.1 °2θ. In a certainembodiment, described herein the crystalline psilocybin Polymorph A′comprising at least 4 peaks of (±0.1 °2θ) of Table 2 but absent orsubstantially absent of a peak at 17.5 °2θ±0.1 °2θ. In a certainembodiment, described herein the crystalline psilocybin Polymorph A′comprises at least 5 peaks of (±0.1 °2θ) of Table 2 but absent orsubstantially absent of a peak at 17.5 °2θ±0.1 °2θ. In a certainembodiment, described herein the crystalline psilocybin Polymorph A′comprises at least 6 peaks of (±0.1 °2θ) of Table 2 but absent orsubstantially absent of a peak at 17.5 °2θ±0.1 °2θ. In a certainembodiment, described herein the crystalline psilocybin Polymorph A′comprises at least 8 peaks of (±0.1 °2θ) of Table 2 but absent orsubstantially absent of a peak at 17.5 °2θ±0.1 °2θ. In a certainembodiment, described herein the crystalline psilocybin Polymorph A′comprises at least 10 peaks of (±0.1 °2θ) of Table 2 but absent orsubstantially absent of a peak at 17.5 °2θ±0.1 °2θ. In a certainembodiment, described herein the crystalline psilocybin Polymorph A′comprises at least 15 peaks of (±0.1 °2θ) of Table 2 but absent orsubstantially absent of a peak at 17.5 °2θ±0.1 °2θ. In a certainembodiment, described herein the crystalline psilocybin Polymorph A′comprises at least 20 peaks of (±0.1 °2θ) of Table 2 but absent orsubstantially absent of a peak at 17.5 °2θ±0.1 °2θ. In a certainembodiment, described herein the crystalline psilocybin Polymorph A′comprises at least 25 peaks of (±0.1 °2θ) of Table 2 but absent orsubstantially absent of a peak at 17.5 °2θ±0.1 °2θ.

TABLE 2 XRPD peak positions for Polymorph A′ Position [°2 Th.] RelativeIntensity [%] 5.5 4.89 10.1 4.09 11.5 22.05 12.0 22.77 14.5 100 14.911.29 17.5 1.08 18.7 2.44 19.4 23.02 19.6 33.7 20.3 17.01 21.1 12.0821.6 8.51 22.2 15.54 22.6 8.78 23.1 10.11 24.3 21.83 25.1 4.36 25.8 15.426.3 4.28 26.8 2.86 27.8 5.96 28.6 1.91 29.7 10.56 31.1 7.35 32.6 3.7233.8 1.54

In one embodiment, crystalline psilocybin Polymorph A′ is characterisedby XRPD diffractogram peaks at 11.5, 12.0, and 14.5 °2θ±0.1 °2θ butsubstantially absent of a peak at 17.5 °2θ±0.1 °2θ. In anotherembodiment, crystalline psilocybin Polymorph A′ is further characterisedby at least one additional peak appearing at 19.7, 20.4, 22.2, 24.3, or25.7 °2θ±0.1 °2θ. In another embodiment, crystalline psilocybinPolymorph A′ is further characterised by at least two additional peaksappearing at 19.7, 20.4, 22.2, 24.3, or 25.7 °2θ±0.1 °2θ. In anotherembodiment, crystalline psilocybin Polymorph A′ is furthercharacterised, and distinguished from Polymorph A by the presence of apeak appearing at 10.1 °2θ±0.1 °2θ. In yet a further embodiment,crystalline psilocybin Polymorph A′ exhibits an XRPD diffractogramsubstantially the same as the XRPD diffractogram shown in FIG. 7 b.

In one embodiment, crystalline psilocybin Polymorph A′ is characterisedby XRPD diffractogram peaks at 14.5 and 17.5 °2θ±0.1 °2θ wherein theintensity of the peak at 17.5 °2θ is less than 5% of the intensity ofthe peak at 14.5 °2θ, such as less than 4%, such as less than 3%, suchas at less than 2%, such as less than 1%, or such as about 1%.

In one embodiment, crystalline psilocybin Polymorph A′ is characterisedby XRPD diffractogram peaks at 10.1 and 14.5 °2θ±0.1 °2θ wherein theintensity of the peak at 10.1 °2θ is at least 1% of the intensity of thepeak at 14.5 °2θ, such as at least than 2%, such as at least than 3%, orsuch as about 4%.

In one embodiment, crystalline psilocybin Polymorph A′ is characterisedby an endothermic event in a DSC thermogram having an onset temperatureof between 205 and 220° C., such as between 210 and 220° C., such asbetween 210 and 218° C., or such as between 210 and 216° C. In anotherembodiment, crystalline psilocybin Polymorph A′ is further characterisedby an endothermic event in the DSC thermogram having an onsettemperature of between 145 and 165° C., such as between 145 and 160° C.,or such as between 145 and 155° C. In another embodiment, crystallinepsilocybin Polymorph A′ is characterised by an endothermic event havingan onset temperature of between 205 and 220° C., such as between 210 and220° C., such as between 210 and 218° C., or such as between 210 and216° C., and an endothermic event having an onset temperature of between145 and 165° C., such as between 145 and 160° C., or such as between 145and 155° C., in a DSC thermogram. In yet another embodiment, crystallinepsilocybin Polymorph A′ exhibits a DSC thermogram substantially the sameas the DSC thermogram in FIG. 8 b.

In another embodiment, crystalline psilocybin Polymorph A′ ischaracterised by having a water content of <0.5% w/w, such as <0.4% w/w,such as <0.3% w/w, such as <0.2% w/w, or such as <0.1% w/w. The skilledperson would know of methods to determine the water content of acompound, for example Karl Fischer Titration. In one embodiment,crystalline psilocybin Polymorph A′ is characterised by having <0.5% w/wloss, such as <0.4% w/w, such as <0.3% w/w, such as <0.2% w/w, such as<0.1% w/w, in the TGA thermogram between ambient temperature, such as25° C., and 200° C. In one embodiment, crystalline psilocybin PolymorphA′ loses less than 2% by weight in a loss on drying test, such as lessthan 1% by weight, such as less than 0.5% by weight. The loss on dryingtest is performed at 70° C.

In one embodiment, crystalline psilocybin Polymorph A′ is a highly purecrystalline form of Polymorph A′, for example, psilocybin comprises atleast 90% by weight, such as 95%, such as 99%, such as 99.5% ofPolymorph A′.

In one embodiment, crystalline psilocybin Polymorph A's is a white tooff white solid.

In another embodiment, crystalline psilocybin Polymorph A′ is chemicallypure, for example the psilocybin has a chemical purity of greater than97%, more preferably still greater than 98%, and most preferably greaterthan 99% by HPLC. In one embodiment, crystalline psilocybin Polymorph A′has no single impurity of greater than 1%, more preferably less than0.5%, including phosphoric acid as measured by ³¹P NMR, and psilocin asmeasured by HPLC. In one embodiment, crystalline psilocybin Polymorph A′has a chemical purity of greater than 97 area %, more preferably stillgreater than 98 area %, and most preferably greater than 99 area % byHPLC. In one embodiment, crystalline psilocybin Polymorph A′ has nosingle impurity greater than 1 area %, more preferably less than 0.5area % as measured by HPLC. In one embodiment, crystalline psilocybinPolymorph A′ does not contain psilocin at a level greater than 1 area %,more preferably less than 0.5 area % as measured by HPLC. In oneembodiment, crystalline psilocybin Polymorph A′ does not containphosphoric acid at a level greater than 1 weight %, more preferably lessthan 0.5 weight % as measured by ³¹P NMR. In one embodiment, crystallinepsilocybin Polymorph A′ has a chemical assay of at least 95 weight %,such as at least 96 weight %, or such as at least 98 weight %.

XRPD diffractograms and XRPD peak positions are acquired using Cu Kαradiation.

DSC and TGA thermograms are acquired using a heating rate of 20° C./min.

In one embodiment, there is provided high purity crystalline psilocybin,Polymorph A or Polymorph A′ (12A or 12A′), exhibiting an XRPDdiffractogram as substantially illustrated in FIG. 7a or 7 b and a DSCthermograph as substantially illustrated in FIG. 8a or 8 b or a mixturethereof.

Preferably the crystalline psilocybin Polymorph A (12A) exhibits an XRPDdiffractogram as illustrated in FIG. 7a and a DSC thermograph asillustrated in FIG. 8 a.

Preferably the crystalline psilocybin Polymorph A′ (12A′) exhibits anXRPD diffractogram as substantially illustrated in FIG. 7b and a DSCthermograph as substantially illustrated in FIG. 8 b.

Preferably the high purity crystalline psilocybin Polymorph A (12A) ischaracterised by a XRPD diffractogram as substantially illustrated inFIG. 7a and a DSC thermograph as substantially illustrated in FIG. 8 a.

Preferably the high purity crystalline psilocybin Polymorph A (12A′) ischaracterised by a XRPD diffractogram as illustrated in FIG. 7b and aDSC thermograph as illustrated in FIG. 8 b.

Polymorph A (including its isostructural variant Polymorph A′) (FIGS. 7aand 7b ) differs from Polymorph B (FIG. 7c ), the Hydrate A (FIG. 7d )and the ethanol solvate (FIG. 7e : Solvate A), and the relationshipbetween some of the different forms is illustrated in FIG. 9.

The crystalline psilocybin Polymorph A or Polymorph A′, is a white tooff white solid, and/or has a chemical purity of greater than 97%, morepreferably still greater than 98%, and most preferably greater than 99%by HPLC, and has no single impurity of greater than 1%, more preferablyless than 0.5%, including phosphoric acid as measured by ³¹P NMR, andpsilocin as measured by HPLC. In one embodiment, there is provided highpurity crystalline psilocybin, Polymorph A or Polymorph A′. In oneembodiment, crystalline psilocybin, Polymorph A or Polymorph A′, has achemical purity of greater than 97 area %, more preferably still greaterthan 98 area %, and most preferably greater than 99 area % by HPLC. Inone embodiment, crystalline psilocybin, Polymorph A or Polymorph A′, hasno single impurity greater than 1 area %, more preferably less than 0.5area % as measured by HPLC. In one embodiment, crystalline psilocybin,Polymorph A or Polymorph A′, does not contain psilocin at a levelgreater than 1 area %, more preferably less than 0.5 area % as measuredby HPLC. In one embodiment, crystalline psilocybin, Polymorph A orPolymorph A′, does not contain phosphoric acid at a level greater than 1weight %, more preferably less than 0.5 weight % as measured by ³¹P NMR.In one embodiment, crystalline psilocybin, Polymorph A or Polymorph A′,has a chemical assay of at least 95 weight %, such as at least 96 weight%, or such as at least 98 weight %.

The heating of Polymorph A or A′ results in an endothermic event havingan onset temperature of circa 150° C. corresponding to solid-solidtransition of Polymorph A or Polymorph A′ to Polymorph B. Continuedheating of the resulting solid, i.e., Polymorph B, results in a secondendothermic event corresponding to a melting point having an onsettemperature of between 205 and 220° C. (see FIGS. 8a and 8b ).

In accordance with another independent aspect of the present inventionthere is provided a crystalline form of psilocybin, Hydrate A,characterised by one or more of:

-   a. peaks in an XRPD diffractogram at 8.9, 12.6 and 13.8 °2θ±0.1 °2θ;-   b. peaks in an XRPD diffractogram at 8.9, 12.6 and 13.8 °2θ±0.1 °2θ,    further characterised by at least one further peak at 6.5, 12.2,    19.4, 20.4 or 20.8 °2θ±0.1 °2θ;-   c. an XRPD diffractogram as substantially illustrated in FIG. 7d ;    or-   d. an endothermic event in a DSC thermogram having an onset    temperature of between 205 and 220° C. substantially as illustrated    in FIG. 8 d.

In one embodiment, psilocybin Hydrate A exhibits an XRPD diffractogramcharacterised by the diffractogram summarised in Table 3. In oneembodiment, described herein the crystalline psilocybin Hydrate Acomprises at least 3 peaks of (±0.1 °2θ) of Table 3. In a certainembodiment, described herein the crystalline psilocybin Hydrate Acomprises at least 4 peaks of (±0.1 °2θ) of Table 3. In a certainembodiment, described herein the crystalline psilocybin Hydrate Acomprises at least 5 peaks of (±0.1 °2θ) of Table 3. In a certainembodiment, described herein the crystalline psilocybin Hydrate Acomprises at least 8 peaks of (±0.1 °2θ) of Table 3. In a certainembodiment, described herein the crystalline psilocybin Hydrate Acomprises at least 10 peaks of (±0.1 °2θ) of Table 3.

TABLE 3 XRPD peak positions for Hydrate A Position [°2 Th.] RelativeIntensity [%] 5.6 14.4 6.5 18.84 8.9 100 12.2 11.51 12.6 18.65 13.844.22 16.2 21.22 18.9 6.62 19.4 38.68 20.4 21.32 20.8 19.73 21.5 20.7522.3 12.8 22.5 19.38 23.1 47.53 23.5 25.79 24.3 5.62 24.8 14.62 25.45.27 26.9 6.53 27.9 7.82 28.4 5.78 29.0 5.09 29.7 4.83 32.1 8.27 32.84.81 33.4 3.74 34.2 5.96

In one embodiment, crystalline psilocybin Hydrate A is characterised byXRPD diffractogram peaks at 8.9, 12.6 and 13.8 °2θ±0.1 °2θ. In anotherembodiment, crystalline psilocybin Hydrate A is further characterised byat least one peak appearing at 6.5, 12.2, 19.4, 20.4 or 20.8 °2θ±0.1°2θ. In another embodiment, crystalline psilocybin Hydrate A is furthercharacterised by at least two peaks appearing at 6.5, 12.2, 19.4, 20.4or 20.8 °2θ±0.1 °2θ. In yet a further embodiment, crystalline psilocybinHydrate A exhibits an XRPD diffractogram substantially the same as theXRPD diffractogram shown in FIG. 7 d.

In one embodiment, crystalline psilocybin Hydrate A is characterised byan endothermic event in a DSC thermogram having an onset temperature ofbetween 205 and 220° C., such as between 210 and 220° C., such asbetween 210 and 218° C., or such as between 210 and 216° C. In anotherembodiment, crystalline psilocybin Hydrate A is further characterised byan endothermic event in the DSC thermogram having an onset temperatureof between 85 and 105° C., or such as between 90 and 100° C. In anotherembodiment, crystalline psilocybin Hydrate A is characterised by anendothermic event having an onset temperature of between 205 and 220°C., such as between 210 and 220° C., such as between 210 and 218° C., orsuch as between 210 and 216° C., and an endothermic event having anonset temperature of between 85 and 105° C., or such as between 90 and100° C., in a DSC thermogram. In yet another embodiment, crystallinepsilocybin Hydrate A exhibits a DSC thermogram substantially the same asthe DSC thermogram in FIG. 8 d.

In another embodiment, crystalline psilocybin Hydrate A is characterisedby having a water content of between 10 and 18%, such as between 12 and16%, or such as about 13%.

The skilled person would know of methods to determine the water contentof a compound, for example Karl Fischer Titration. In one embodiment,crystalline psilocybin Hydrate A is characterised by having a weightloss in the TGA thermogram of between 10 and 18%, such as between 12 and16%, or such as about 13%, between ambient temperature, such as about25° C., and 120-C.

In one embodiment, crystalline psilocybin Hydrate A is a highly purecrystalline form of Hydrate A, for example, psilocybin comprises atleast 90% by weight, such as 95%, such as 99%, such as 99.5% of HydrateA.

In accordance with another independent aspect of the present inventionthere is provided a crystalline form of psilocybin, Polymorph B,characterised by one or more of:

-   a. peaks in an XRPD diffractogram at 11.1, 11.8 and 14.3 °2θ±0.1    °2θ;-   b. peaks in an XRPD diffractogram at 11.1, 11.8 and 14.3 °2θ±0.1    °2θ, further characterised by at least one further peak at 14.9,    15.4, 19.3, 20.0 or 20.6 °2θ±0.1 °2θ;-   c. an XRPD diffractogram as substantially illustrated in FIG. 7c ;    or-   d. an endothermic event in a DSC thermogram having an onset    temperature of between 205 and 220° C. substantially as illustrated    in FIG. 8 c.

In one embodiment, psilocybin Polymorph B exhibits an XRPD diffractogramcharacterised by the diffractogram summarised in Table 4. In oneembodiment, described herein the crystalline psilocybin Polymorph Bcomprises at least 3 peaks of (±0.1 °2θ) of Table 4. In a certainembodiment, described herein the crystalline psilocybin Polymorph Bcomprises at least 4 peaks of (±0.1 °2θ) of Table 4. In a certainembodiment, described herein the crystalline psilocybin Polymorph Bcomprises at least 5 peaks of (±0.1 °2θ) of Table 4. In a certainembodiment, described herein the crystalline psilocybin Polymorph Bcomprising at least 8 peaks of (±0.1 °2θ) of Table 4. In a certainembodiment, described herein the crystalline psilocybin Polymorph Bcomprises at least 10 peaks of (±0.1 °2θ) of Table 4.

TABLE 4 XRPD peak positions for Polymorph B Position Relative Intensity[°2 Th.] [%] 5.5 21.33 11.1 36.91 11.8 100.00 12.5 12.73 14.3 70.23 14.950.01 15.4 23.67 17.1 51.58 17.4 91.25 18.0 12.61 19.3 39.33 20.0 76.6120.6 50.26 21.5 20.77 22.3 40.19 23.9 13.32 24.3 16.03 25.3 32.94 28.37.60 28.9 17.89 29.3 8.96 31.3 6.57 32.2 6.90 33.8 2.37

In one embodiment, crystalline psilocybin Polymorph B is characterisedby XRPD diffractogram peaks at 11.1, 11.8 and 14.3 °2θ±0.1 °2θ. Inanother embodiment, crystalline psilocybin Polymorph B is furthercharacterised by at least one peak appearing at 14.9, 15.4, 19.3, 20.0or 20.6 °2θ±0.1 °2θ. In another embodiment, crystalline psilocybinPolymorph B is further characterised by at least two peaks appearing at14.9, 15.4, 19.3, 20.0 or 20.6 °2θ±0.1 °2θ. In yet a further embodiment,crystalline psilocybin Polymorph B exhibits an XRPD diffractogramsubstantially the same as the XRPD diffractogram shown in FIG. 7 c.

In one embodiment, crystalline psilocybin Polymorph B is characterisedby an endothermic event in a DSC thermogram having an onset temperatureof between 205 and 220° C., such as between 210 and 220° C., such asbetween 210 and 218° C., or such as between 210 and 216° C. In yetanother embodiment, crystalline psilocybin Polymorph B exhibits a DSCthermogram substantially the same as the DSC thermogram in FIG. 8 c.

In another embodiment, crystalline psilocybin Polymorph B ischaracterised by having a water content of <0.5% w/w, such as <0.4% w/w,such as <0.3% w/w, such as <0.2% w/w, or such as <0.1% w/w. The skilledperson would know of methods to determine the water content of acompound, for example Karl Fischer Titration. In one embodiment,crystalline psilocybin Polymorph B is characterised by having <0.5% w/wloss, such as <0.4% w/w, such as <0.3% w/w, such as <0.2% w/w, such as<0.1% w/w, in the TGA thermogram between ambient temperature, such asabout 25° C., and 200° C. In one embodiment, crystalline psilocybinPolymorph B loses less than 2% by weight in a loss on drying test, suchas less than 1% by weight, such as less than 0.5% by weight. The loss ondrying test is performed at 70° C.

In one embodiment, crystalline psilocybin Polymorph B is a highly purecrystalline form of Polymorph B, for example, psilocybin comprises atleast 90% by weight, such as 95%, such as 99%, such as 99.5% ofPolymorph B.

In another embodiment, crystalline psilocybin Polymorph B is chemicallypure, for example the psilocybin has a chemical purity of greater than97%, such as greater than 98%, or such as greater than 99% by HPLC. Inone embodiment, crystalline psilocybin Polymorph B has no singleimpurity of greater than 1%, more preferably less than 0.5%, includingphosphoric acid as measured by ³¹P NMR, and psilocin as measured byHPLC. In one embodiment, crystalline psilocybin Polymorph B has achemical purity of greater than 97 area %, more preferably still greaterthan 98 area %, and most preferably greater than 99 area % by HPLC. Inone embodiment, crystalline psilocybin Polymorph B has no singleimpurity greater than 1 area %, more preferably less than 0.5 area % asmeasured by HPLC.

In one embodiment, crystalline psilocybin Polymorph B does not containpsilocin at a level greater than 1 area %, more preferably less than 0.5area % as measured by HPLC. In one embodiment, crystalline psilocybinPolymorph B does not contain phosphoric acid at a level greater than 1weight %, more preferably less than 0.5 weight % as measured by ³¹P NMR.In one embodiment, crystalline psilocybin Polymorph B has a chemicalassay of at least 95 weight %, such as at least 96 weight %, or such asat least 98 weight %.

The psilocybin of the invention in the form Polymorph A or A′ has thegeneral properties illustrated in Table 5 below:

TABLE 5 Appearance: White to off white solid Major endothermic event in210-215° C. DSC (onset temperature) (corresponding to a melt):Hygroscopicity: Psilocybin forms Hydrate A at high humidity and whenadded to water but the water of hydration is lost rapidly on drying. Theanhydrous form is therefore being developed. Crystalline form: AnhydrousPolymorph A and/ or A′ pKa (calculated): 1.74, 6.71, 9.75 Solubilityapprox. 15 mg/ml in Water

The psilocybin conforms to the spectra as set out in Table 6 below andillustrated in the spectra of FIGS. 10-13.

TABLE 6 Technique Conclusions Proton (¹H) and Carbon (¹³C) NMRAssignment of the proton (FIG. 10) and carbon spectra (FIG. 11) areconcordant with Psilocybin. FT-Infrared Spectroscopy (FT-IR) Assignmentof the FT-IR spectrum (FIG. 12) is concordant with Psilocybin. MassSpectroscopy (MS) Assignment of the mass spectrum (FIG. 13) isconcordant with Psilocybin.

The high purity is attained by careful control of reaction conditions toensure that potential organic impurities are significantly reduced.

Known and potential impurities in Psilocybin are shown in Table 7 below:

TABLE 7 Relative Retention Time Impurity (RRT) Structure Origin Psilocin1.65

Starting material (stage 3). Also generated by hydrolysis of Psilocybin.Only significant impurity observed in psilocybin batches. Stage 4A

Initial product formed in the stage 4 reaction. Converts to stage 4 onstirring in THF. Converts to Psilocybin in stage 5. Stage 4 2.74

Intermediate N-Denzylated stage 4

Identified by MS in Stage 4. Converts to Psilocybin in stage 5. Stage 4Anhydride Impurity

Identified by MS in Stage 4. Converts to Stage 5 Pyrophosphoric acidimpurity in stage 5. Stage 5 Pyrophosphoric acid impurity

Identified by MS Formed from the stage 4 anhydride. Removed in the stage6 re- crystallisation by a combination of hydrolysis to Psilocybin andincreased solubility due to the extra phosphate group. Stage 5(intermediates) 1.89 and 2.45

2 Intermediates are formed during the hydrogenation. These subsequentlyconvert to product (structures based on chemistry). Monitored andcontrolled in stage 5 reaction.

Similarly, the careful processing ensures solvent levels are kept tobelow levels as indicated in Table 8.

TABLE 8 Chemical Stage solvent is Solvent Controlled to used in Methanol3000 ppm Stage 5 Ethanol 5000 ppm Stage 5 THF  720 ppm Stage 4 Toluene 890 ppm Generated as a by-product in stage 5

Through careful selection of operating methodology the psilocybin drugsubstance of the invention meets the acceptance criteria set out inTable 9 below:

TABLE 9 Quality attribute Acceptance criteria Test method 1. AppearanceFor information only. Visual 2. Identity by ¹H NMR Compares well with ¹HNMR reference. FIG. 10 3. Identity by ¹³C NMR Compares well with ¹³C NMRreference. FIG. 11 4. Identity by MS Compares well with MS reference.FIG. 12 5. Identity by FT-IR Compares well with FT-IR reference. FIG. 136. Loss on drying NMT 2% w/w European Pharmacopoeia 2.2.32 7. Residue onIgnition NMT 0.5% w/w US Pharmacopoeia <281> 8. Chemical purity NLT 97area % HPLC 9. Drug Related No single impurity NMT HPLC Impurities 1.0area % 10. Assay (on a dry 95-103 weight % HPLC basis) 11. ResidualSolvent Methanol NMT 3000 ppm HRGC Content Ethanol NMT 5000 ppm THF NMT720 ppm Toluene NMT 890 ppm 12. Phosphoric acid NMT 1% w/w ³¹P NMRcontent 13. Elemental analysis Cd NMT 1.5 ppm US Pharmacopoeia by ICP-MSPb NMT 1.5 ppm <233> As NMT 4.5 ppm Hg NMT 9.0 ppm Co NMT 15 ppm V NMT30 ppm Ni NMT 60 ppm Li NMT 165 ppm Pd NMT 30 ppm 14. PolymorphismConforms to reference XRPD FIG. 7a 15. Melting Point Report result DSCFIG. 8a Abbreviations used in table: NMT = not more than, NLT = not lessthan.

The methodology used to verify the purity is provided in the detaileddescription.

In fact, the criteria 6-13 are far exceeded in practice, as noted inTable 10 below:

TABLE 10 Acceptance Quality attribute criteria Typically Test method 1.Loss on drying Typically less European than 1% w/w Pharmacopoeia 2.2.322. Residue on Ignition Typically less US Pharmaco- than 0.2% w/w poeia<281> 3. Chemical purity Typically NLT HPLC 99% 4. Drug Related Nosingle RRT 1.49: 0.06% HPLC Impurity impurity RRT 1.69 NMT 1.0%(Psilocin): 0.39% RRT 1.70: 0.05% Others LT 0.05%: 0.22% 5. Assay (on adry 95-103 98.65% HPLC basis) 6. Residual Solvent Methanol NMT 5 ppmHRGC Content NMT NMT 10 ppm 3000 ppm NMT 5 ppm Ethanol NMT 5 ppm NMT5000 ppm THF NMT 720 ppm Toluene NMT 890 ppm 7. Phosphoric acid NMT 1%w/w 0.2% ³¹P NMR content Absence of phosphoric acid (H₃PO₄) which comesat approx. 0 ppm 8. Elemental analysis Cd NMT LT 0.5 ppm US Pharmaco- byICP-MS 1.5 ppm LT 0.5 ppm poeia <233> Pb NMT LT 1 ppm 1.5 ppm LT 1 ppmAs NMT LT 5 ppm 4.5 ppm LT 20 ppm Hg NMT LT 10 ppm 9.0 ppm LT 20 ppm CoNMT LT 5 ppm 15 ppm V NMT 30 ppm Ni NMT 60 ppm Li NMT 165 ppm Pd NMT 30ppm Abbreviations used in table: NMT = not more than, LT = less than.

Thus, crystalline psilocybin, in the form Polymorph A or Polymorph A′,has spectra that conform with Proton (¹H) and Carbon (¹³C) NMR,FT-Infrared Spectroscopy (FT-IR), and Mass Spectroscopy (MS)—FIGS.10-13.

It also conforms to any of the criteria specified in Table 9 or Table10.

In accordance with a second aspect of the present invention there isprovided a batch of crystalline psilocybin, in the form Polymorph A orPolymorph A′ according to the first aspect of the present invention. Inone embodiment, there is provided a batch of crystalline psilocybin,Polymorph A or Polymorph A′, comprising at least 10 g, more preferablyat least 100 g, and most preferably at least 250 g. In one embodiment,there is provided a batch of crystalline psilocybin, Polymorph A orPolymorph A′, comprising at least 10 g, more preferably at least 100 g,and most preferably at least 250 g. In one embodiment, there is provideda batch of high purity psilocybin comprising at least 10 g, morepreferably at least 100 g, and most preferably at least 250 g. In oneembodiment, there is provided a batch of high purity psilocybinPolymorph A comprising at least 10 g, more preferably at least 100 g,and most preferably at least 250 g. In one embodiment, there is provideda batch of high purity psilocybin Polymorph A′ comprising at least 10 g,more preferably at least 100 g, and most preferably at least 250 g.

Alternatively, and independently, the crystalline psilocybin may takethe form of Hydrate A or Polymorph B.

In accordance with a third aspect of the present invention there isprovided a pharmaceutical formulation comprising crystalline psilocybinand one or more excipients.

In one embodiment, there is provided a pharmaceutical formulationcomprising high purity psilocybin and one or more excipients. In anotherembodiment, there is provided a pharmaceutical formulation comprisingcrystalline psilocybin Polymorph A and one or more excipients. Inanother embodiment, there is provided a pharmaceutical formulationcomprising crystalline psilocybin Polymorph A′ and one or moreexcipients. In another embodiment, there is provided a pharmaceuticalformulation comprising high purity crystalline psilocybin, Polymorph Aor Polymorph A′, and one or more excipients. In another embodiment,there is provided a pharmaceutical formulation comprising high puritycrystalline psilocybin Polymorph A and one or more excipients. Inanother embodiment, there is provided a pharmaceutical formulationcomprising high purity crystalline psilocybin Polymorph A′ and one ormore excipients.

Alternatively, and independently, the crystalline psilocybin in theformulation may take the form of Hydrate A or Polymorph B.

Preferred pharmaceutical excipients for an oral formulation include:diluents, such as, microcrystalline cellulose, starch, mannitol, calciumhydrogen phosphate anhydrous or co-mixtures of silicon dioxide, calciumcarbonate, microcrystalline cellulose and talc; disintegrants, such as,sodium starch glycolate or croscarmellose sodium; binders, such as,povidone, co-povidone or hydroxyl propyl cellulose; lubricants, such as,magnesium stearate or sodium stearyl fumurate; glidants, such as,colloidal silicon dioxide; and film coats, such as, Opadry II white orPVA based brown Opadry II.

Psilocybin is a difficult active to formulate for a number of reasons.Firstly it has poor flow characteristics, and secondly it is used inrelatively low doses which combination makes it challenging to ensurecontent uniformity in tabletting.

A good blend will have an Acceptance Value, AV value of less than 15,and more preferably less than 10.

It will also have a % Label claim of greater than 90% more preferablygreater than 94%.

Between them these parameters indicate consistent dosing of thepsilocybin between tablets.

For most pharmaceutical tablets, standard excipients, particularlyfillers, can be used. However, in the course of formulating psilocybintablets, applicant found that in order to achieve a satisfactoryproduct, a non-standard filler was preferred.

In this regard a functional filler was selected. The functional fillerwas a silicified filler, preferably a silicified microcrystallinecellulose. The preferred forms comprises high compactability grades witha particle size range of from about 45 to 150 microns.

In fact a mixture of two functional fillers having different particlesize ranges may be used with the wt percentages of the two favouring thelarger sized particles.

In one embodiment the silicified microcrystalline filler may comprise afirst filler, having a particle size range of from about 45 to 80microns in an amount of up to 30%, more preferably up to 20%, morepreferably still up to 15% or less and a second filler, having aparticle size range of from about 90 to 150 microns, in an amount of upto 70%, more preferably up to 80, and more preferably still up to 85% ormore, by weight.

The formulation may further comprise or consist of a disintegrant,preferably sodium starch glycolate, a glidant, preferably colloidalsilicon dioxide and a lubricant, preferably sodium stearyl fumarate.

Further details of formulation development are given in Example 12.

It should be noted that the formulations may comprise psilocybin in anyform, not only the preferred polymorphic forms disclosed.

Studerus et al (2011) J Psychopharmacol 25(11) 1434-1452 classified oraldoses of psilocybin as follows: Very low doses at 0.045 mg·kg; low dosesbetween 0.115-0.125 mg/kg, medium doses between 0.115-0.260 mg/kg, andhigh doses at 0.315 mg/kg.

The psilocybin would typically be present in a formulated dose in anamount of from 0.01 mg/kg to 1 mg/kg. A typical human dose (for an adultweighing 60-80 kg) would equate to a dose of somewhere between 0.60 mgand 80 mg. In one embodiment, between 2 and 50 mg of crystallinepsilocybin, most preferably Polymorph A or Polymorph A′, is present in aformulated dose, such as between 2 and 40 mg, such as between 2 and 10mg, such as 5 mg, such as between 5 and 30 mg, such as between 5 and 15mg, such as 10 mg, such as between 20 and 30 mg, or such as 25 mg. Inone embodiment, between 2 and 50 mg of crystalline psilocybin,particularly Polymorph A, is present in a formulated dose, such asbetween 2 and 40 mg, such as between 2 and 10 mg, such as 5 mg, such asbetween 5 and 30 mg, such as between 5 and 15 mg, such as 10 mg, such asbetween 20 and 30 mg, or such as 25 mg. In one embodiment, between 2 and50 mg of crystalline psilocybin, particularly Polymorph A′ is present ina formulated dose, such as between 2 and 40 mg, such as between 2 and 10mg, such as 5 mg, such as between 5 and 30 mg, such as between 5 and 15mg, such as 10 mg, such as between 20 and 30 mg, or such as 25 mg.

Favoured adult oral doses are likely to be in the range 1 mg to 40 mg,preferably 2 to 30 mg, more preferably 15 to 30 mg, for example 5 mg, 10mg or 25 mg. Micro-dosing, typically at about a tenth of these doses, isalso possible with micro dose formulations typically lying within therange 0.05 mg to 2.5 mg.

A preferred pharmaceutical formulation is an oral dosage form.

The oral dosage form may be a tablet or a capsule.

For a tablet it is necessary to be able to accurately disperse theactive. This is challenging due to the low doses and the hygroscopic andsticky nature of the active which limits its flowability.

The psilocybin will be present together with one or more excipients.Preferred excipients include microcrystalline cellulose and starch, moreparticularly still a silicified microcrystalline cellulose.

In accordance with a fourth aspect of the present invention there isprovided the crystalline psilocybin in the form Polymorph A or PolymorphA′ according to the first aspect of the present invention for use inmedicine. In one embodiment, there is provided crystalline psilocybinPolymorph A for use in medicine. In one embodiment, there is providedcrystalline psilocybin Polymorph A′ for use in medicine. In oneembodiment, there is provided a high purity crystalline psilocybinPolymorph A for use in medicine. In one embodiment, there is provided ahigh purity crystalline psilocybin Polymorph A′ for use in medicine.

Alternatively, and independently, the crystalline psilocybin may takethe form of Hydrate A or Polymorph B.

In accordance with a fifth aspect of the present invention there isprovided crystalline psilocybin in the form Polymorph A or Polymorph A′of the first aspect of the present invention for use in treating centralnervous disorders.

Alternatively, and independently, the crystalline psilocybin may takethe form of Hydrate A or Polymorph B.

In one embodiment, there is provided crystalline psilocybin, Polymorph Aor Polymorph A′, for use in treating depression. In one embodiment,there is provided crystalline psilocybin, Polymorph A or Polymorph A′,for use in treating drug resistant depression. In one embodiment, thereis provided crystalline psilocybin Polymorph A for use in treating drugresistant depression. In one embodiment, there is provided crystallinepsilocybin Polymorph A′ for use in treating drug resistant depression.In one embodiment, there is provided a high purity crystallinepsilocybin Polymorph A for use in treating drug resistant depression. Inone embodiment, there is provided a high purity crystalline psilocybinPolymorph A′ for use in treating drug resistant depression.

Other conditions that may be treated include: anxiety disorders,including anxiety in advanced stage illness e.g. cancer as well asGeneralized Anxiety Disorder, Depression including Major DepressiveDisorder, Cluster Headaches, Obsessive Compulsive Disorder, PersonalityDisorders including Conduct Disorder, Drug Disorders including: alcoholdependence, nicotine dependence, opioid dependence, cocaine dependenceand other addictions including Gambling Disorder, Eating Disorder andBody Dysmorphic Disorder. A still further condition is the treatment ofpain.

In accordance with a sixth aspect of the present invention there isprovided a method of treating central nervous disorders comprisingadministering to a subject in need thereof an effective dose ofcrystalline psilocybin in the form Polymorph A or Polymorph A′ accordingto the first aspect of the present invention.

In one embodiment, there is provided a method of treating depressioncomprising administering to a subject in need thereof an effective doseof crystalline psilocybin in the form of Polymorph A or Polymorph A′. Inone embodiment, there is provided a method of treating drug resistantdepression comprising administering to a subject in need thereof aneffective dose of crystalline psilocybin in the form Polymorph A orPolymorph A′. In one embodiment, there is provided a method of treatingdrug resistant depression comprising administering to a subject in needthereof an effective dose of psilocybin Polymorph A. In one embodiment,there is provided a method of treating drug resistant depressioncomprising administering to a subject in need thereof an effective doseof psilocybin Polymorph A′. In one embodiment, there is provided amethod of treating drug resistant depression comprising administering toa subject in need thereof an effective dose of a high purity crystallinepsilocybin Polymorph A. In one embodiment, there is provided a method oftreating drug resistant depression comprising administering to a subjectin need thereof an effective dose of a high purity crystallinepsilocybin Polymorph A′.

Alternatively, and independently, the crystalline psilocybin may takethe form of Hydrate A or Polymorph B.

To produce the psilocybin of the invention the psilocybin wascrystallised from water in a controlled manner.

According to a seventh aspect of the present invention there is provideda method for large scale manufacture of psilocybin characterised in thatthe method comprises subjecting psilocybin to a water crystallizationstep, with controlled drying, to produce crystalline psilocybinPolymorph A according to the first aspect of the present invention.

In one embodiment, there is provided a method for large scalemanufacture of psilocybin characterised in that the method comprisessubjecting psilocybin to a water crystallization step, with controlleddrying, to produced crystalline psilocybin Polymorph A with an XRPDdiffractogram as illustrated in FIG. 7a and a DSC and TGA thermograph asillustrated in FIG. 8a . In one embodiment, there is provided a methodfor large scale manufacture of psilocybin characterised in that themethod comprises subjecting psilocybin to a water crystallization step,with controlled drying, to produce a high purity crystallinepsilocybin—Polymorph A with an XRPD diffractogram as illustrated in FIG.7a and a DSC thermograph as illustrated in FIG. 8 a.

Preferably Polymorph A is an isostructural variant with an XRPDdiffractogram as illustrated in FIG. 7a and a DSC thermograph asillustrated in FIG. 8 a.

More preferably the psilocybin is recrystallized in typically about10-20 volumes of water, heated with agitation to a temperature of atleast 70° C., polish filtered with a suitable cut off (typically, below5 μm), seeded at a temperature of about 70° C., and cooled in acontrolled manner to about 5° C. over a period of more than 2 hours.

More preferably the method comprises controlled cooling which drops thetemperature by about 5° C.-15° C. an hour, more preferably about 10° C.an hour.

Preferably the polish filter step is done through an appropriately sizedfilter such as a 1.2 μm in line filter.

Preferably the agitation is by stirring at about 400-500 rpm, typicallyabout 450 rpm.

Preferably the seed is psilocybin Hydrate A. In one embodiment, 0.1%weight or less of seed is added to the process.

Preferably the crystalline psilocybin is isolated by vacuum filtration.

In one embodiment, the isolated crystals are dried in vacuo at atemperature of at least 30° C., such as between 30 and 50° C., or suchas between 40 and 50° C. In one embodiment, the isolated crystals aredried in vacuo for at least 10 hours, such as between 12 and 18 hours,or such as about 30 hours. In one embodiment, the isolated crystals aredried in vacuo at a temperature of at least 30° C., such as between 30and 50° C., or such as between 40 and 50° C., for at least 10 hours,such as between 12 and 18 hours, or such as about 30 hours. In oneembodiment, the isolated crystals are dried until the isolated crystalslose less than 2% weight in a loss on drying test, such as less than0.5% weight.

Preferably the isolated crystals are washed, several times, in water anddried in vacuo at about 50° C. for at least 12 hours.

The crystals obtained are typically relatively large (range 50 to 200microns) and uniform when viewed under the microscope ×10, asillustrated in FIG. 16 a.

This differs from crystals obtained without controlled cooling which aremuch smaller in size (typically 5 to 50 microns) when viewed under themicroscope ×10, as illustrated in FIG. 16 b.

In accordance with an eighth aspect of the present invention there isprovided Psilocybin according to the first aspect of the presentinvention obtained by the method of crystallisation of the invention.

In accordance with a ninth aspect of the present invention there isprovided a pharmaceutical formulation comprising psilocybin according tothe first aspect of the present invention obtained by the method ofcrystallisation of the invention.

The psilocybin manufactured prior to crystallisation may be producedusing any method: synthetic or biological, e.g. by fermentation orobtained by extraction from mushrooms.

Preferred manufacturing methods use psilocin, or 4 hydroxy-indole, as astarting material.

In accordance with a tenth aspect of the present invention there isprovided a method for large scale manufacture of psilocybin frompsilocin comprising the steps of:

i) Stage 4—Reacting psilocin with tetrabenzylpyrophosphate to formbenzyl 3-[2-(benzyidimethylazaniumyl)ethyl]-1H-indol-4-yl phosphate; and

ii) Stage 5—Reacting benzyl 3-[2-(benzyldimethylazaniumyl)ethyl]-1H-indol-4-yl phosphate with hydrogen to form psilocybin.

In accordance with an eleventh aspect of the present invention there isprovided a method for large scale manufacture of psilocybin from4-hydroxyindole comprising the steps of:

i) Stage 1—Reacting 4-hydroxyindole with acetic anhydride to form1H-indol-4-yl acetate;

ii) Stage 2—Reacting 1H-indol-4-yl acetate with oxalyl chloride anddimethylamine to form3[(dimethylcarbamoyl)carbonyl]-1H-indol-4yl-acetate;

iii) Stage 3—Reacting3[(dimethylcarbamoyl)carbonyl]-1H-indol-4yl-acetate with lithiumaluminium hydride to form psilocin;

iv) Stage 4—Reacting psilocin with tetrabenzylpyrophosphate to formbenzyl 3-[2-(benzyidimethylazaniumyl)ethyl]-1H-indol-4-yl phosphate; and

v) Stage 5—Reacting benzyl 3-[2-(benzyldimethylazaniumyl)ethyl]-1H-indol-4-yl phosphate with hydrogen to form psilocybin.

In accordance with a twelfth aspect of the present invention there isprovided a method for large scale manufacture of psilocybin as per thetenth or eleventh aspect of the present invention further comprising:

vi) Stage 6—a water crystallization step, with controlled drying, toproduce crystalline psilocybin Polymorph A according to the first aspectof the present invention.

In one embodiment, there is provided a method for large scalemanufacture of psilocybin as per the tenth or eleventh aspect of thepresent invention further comprising:

vi) Stage 6—a water crystallization step, with controlled drying, toproduce crystalline psilocybin—Polymorph A with an XRPD diffractogram assubstantially illustrated in FIG. 7a and a DSC thermograph assubstantially illustrated in FIG. 8 a.

In one embodiment, there is provided a method for large scalemanufacture of psilocybin as per the tenth or eleventh aspect of thepresent invention further comprising:

vi) Stage 6—a water crystallization step, with controlled drying, toproduce a high purity crystalline psilocybin—Polymorph A with an XRPDdiffractogram as illustrated in FIG. 7a and a DSC thermograph asillustrated in FIG. 8 a.

Preferably the crystalline psilocybin is Polymorph A.

In developing methodology for the large scale production of psilocin orpsilocybin the Applicant overcame one or more significant problems ateach of Stages 1 to 5, and whilst these problems are considered in thecontext of the large scale production of psilocin or psilocybin eachstep, or rather the way each problem was overcome, are consideredseparate and independent inventions as they have application in themanufacture of other actives be they intermediates to psilocin,psilocybin, or other derivatives, salts, esters or the like whichprovide a prodrug.

Preferably the Stage 4 (i) reaction comprises the use of sodiumhexamethyldisilazide (NaHMDS).

This has the benefits over the use of Butyl lithium in that: i) it iseasier to handle, and ii) it does not introduce lithium into thereaction which causes issues in downstream processing.

Preferably the reaction uses the solvent THF.

This has the benefit that resulting product is obtained in significantlyhigher purity.

Preferably in (i) the reaction is initiated below −50° C.

This has the benefit of reducing the levels of impurities (m/z 295.2observed by LCMS) that will subsequently effect purity downstream.

More preferably still the Stage 4 (ii) step uses THF as the solvent.

This has the benefit of ensuring thickening is avoided and facilitates asimple stir out process for obtaining the product.

Preferably the Stage 4 (ii) step comprises a stir out process to obtainbenzyl 3-[2-(benzyidimethylazaniumyl) ethyl]-1H-indol-4-yl phosphate.

A stir out process has the advantage that the process is simplified andyields are improved.

To ensure the Stage 4 (ii) reaction is run to completion, levels ofIntermediate 4A are monitored, and on completion, the benzyl3-[2-(benzyldimethylazaniumyl) ethyl]-1H-indol-4-yl phosphate isfiltered and oven dried.

This has the advantage that impurities are minimised and a purer productis obtained

Preferably the Stage 5 reaction is monitored for levels of intermediatesby HPLC, using relative retention times (RRT) and completion isdetermined by the intermediates being present at less than 0.2%.

Psilocybin crude (Stage 5 product, (12)) has main stage 5 impuritieswhose relative retention times (RRT) in the HPLC method are about 1.89and 2.45 respectively, and psilocin (RRT 1.66). These impurities areillustrated in Table 7. Typically, psilocybin crude (Stage 5 product(12)) has 0.24 area % of the RRT 1.89 impurity, 0.03 area % of the RRT2.45 impurity and 1.86 area % of psilocin. In addition, thepyrophosphoric acid impurity (RRT 0.31) is present in psilocybin crude,for example at a level of about 2-6 area % by HPLC.

At this level subsequent crystallisation processes can be conducted toprovide substantially pure psilocybin, for example psilocybin having apurity of at least 95 area % by HPLC, such as at least 98 area %, orsuch as at least 99 area %. In one embodiment, the pyrophosphoric acidimpurity (RRT 0.31) is present in the substantially pure psilocybin at alevel of less than 0.3 area % by HPLC, such as less than 0.2 area %, orsuch as less than 0.1 area %.

In addition, during this stage water is added to the reaction tomaintain the psilocybin in solution.

Preferably the catalyst is recovered by filtration.

Preferably in Stage 1 the reaction is conducted in DCM and pyridine.

This has the advantage that flammable solvents are avoided.

Preferably the reaction mixture is washed with citric acid, to give a pHof about 2-3, to remove excess pyridine, and the acid phase is separatedfrom the DCM phase.

This has the advantage that the Intermediate 2A can be isolated,allowing purification away from excess oxalyl chloride.

More preferably the DCM phase is further washed with sodium bicarbonateat about pH 8.

This has the advantage of purer processing.

Preferably the 1H-indol-4-yl acetate is precipitated in heptane.

This aids precipitation and overcomes partial solubility issues.

Preferably magnesium sulphate is used as a drying agent.

Preferably the solvents tert butyl methyl ether (TBME) andtetrahydrofuran (THF) are used.

Preferably the reaction with oxalyl chloride is conducted at about 30°C.-40° C.

This has the advantage that a high reaction rate is ensured givingimproved levels of completion.

Preferably Intermediate 2A is isolated by filtration.

This has the advantage that the intermediate is purified away fromexcess oxalyl chloride.

Preferably in Stage 2, step i the Intermediate 2A is also washed toremove excess oxalyl chloride.

Preferably the Intermediate 2A is washed with TBME.

Preferably a heptane addition is made to precipitate out furtherIntermediate 2A.

Preferably in Stage 2, step ii, dimethyl amine is used in excess.

This has the advantage that a much improved impurity profile and yieldis obtained.

Preferably the pH is maintained at about or above pH 7.

Preferably the reaction is carried out in TBME.

Preferably this stage further comprises a purification step that removesdimethyl amine salts.

This has the advantage that purity is improved.

Preferably this stage comprises a slurry and filtration step.

This has the advantage that handling and purity is improved.

More preferably it comprises slurrying with water and/or IPA, filtering,and drying the isolated3[(dimethylcarbamoyl)carbonyl]-1H-indol-4yl-acetate.

This has the advantage that purity and yields are improved andhydrolysis reduced.

Preferably in Stage 3 the reaction is conducted in the solvent THF.

This has the advantage that a suspension/emulsion is formed withoutthickening.

Preferably the 3[(dimethylcarbamoyl)carbonyl]-1H-indol-4yl-acetate isadded to a solution of LiAlH₄ in THF.

Preferably the reaction is quenched with acetone, followed by citricacid ensuring the mixture remains strongly basic (pH11 or above).

This has the advantage that high yields are obtained.

Preferably the psilocin is filtered and washed in THF and slurried inPrOAc:TBME, filtered, washed in TBME, and dried.

This has the advantage that a high purity product is obtained, forexample, at least 95% pure by HPLC, such as at least 98% pure by HPLC,or such as at least 99% pure by HPLC.

The favoured production method comprises each of Stages 1 to 6 but itwill be appreciated that each of the features of each stage can standalone or be used in combination with any other feature from the same ora different step of the reaction.

Psilocybin of a given form, Polymorph A or Polymorph A′, and psilocybinof such high purity has not previously been obtained, and to Applicantsknowledge their production of Polymorph A and Polymorph A′ particularly(as illustrated in FIGS. 7a & 7 b and 8 a & 8 b) is novel. Indeed, theproduction of large batch quantities of Polymorph A, is new. Aconsequence of the crystallisation methodology of the invention and, inpart, the manufacturing process enable such high chemical purity ofcrystalline Psilocybin to be obtained.

Furthermore, given the unstable nature of the compound they haveobtained a crystalline form which they have shown to be stable, underaccelerated conditions, (described later) for at least 12 months.

Polymorph A and A′ (FIGS. 7a and 7b ) differs from Polymorph B (FIG. 7c), a Hydrate A (FIG. 7d ) and ethanol solvate (FIG. 7e ) and mixture(FIG. 7f (upper)) as will be apparent from their XRPD diffractograms andDSC thermographs—as described hereafter.

The relationship between the different polymorphs is shown in FIG. 9.

Indeed, the size and shape of the crystals are determined by thecrystallisation methodology, and these in turn can affect stability andthe ability to formulate the product.

In a particularly preferred embodiment, the psilocybin is manufacturedthrough a 6-stage process as outlined below:

In accordance with another aspect of the present invention there isprovided a method for manufacture of crystalline psilocybin according tothe first aspect of the present invention, characterised in that themethod comprises subjecting psilocybin to a water crystallization step,with controlled drying, to produce crystalline psilocybin Polymorph A orPolymorph A′ according to the first aspect of the present invention. Inone embodiment, there is provided a method for manufacture ofcrystalline psilocybin according to the first aspect of the presentinvention, characterised in that the method comprises a watercrystallization step, with controlled drying, to produce crystallinepsilocybin—Polymorph A or Polymorph A′ with an XRPD diffractogram assubstantially illustrated in FIG. 7a or FIG. 7b and a DSC thermograph assubstantially illustrated in FIG. 8a or 8 b. In one embodiment, there isprovided a method for manufacture of psilocybin according to the firstaspect of the present invention characterised in that the methodcomprises a water crystallization step, with controlled drying, toproduce a high purity crystalline psilocybin—Polymorph A or Polymorph A′with an XRPD diffractogram as illustrated in FIG. 7a or FIG. 7b and aDSC thermograph as illustrated in FIG. 8a or FIG. 8 b.

Preferably Polymorph A and Polymorph A′ are isostructural variants withXRPD diffractograms as substantially illustrated in FIG. 7a and FIG. 7band DSC thermographs as substantially illustrated in FIG. 8a and FIG. 8b.

More preferably the psilocybin is recrystallized in about 10-20 volumesof water, heated with agitation to a temperature of at least 70° C.,polish filtered with a suitable cut off (typically, below 5 μm), seededat a temperature of about 70° C., and cooled in a controlled manner toabout 5° C. over a period of more than 2 hours.

More preferably the method comprises controlled cooling which drops thetemperature by about 5° C.-15° C. an hour, more preferably about 10° C.an hour.

Preferably the polish filter step is done through an appropriately sizedfilter such as a 1.2 μm or a 0.45 μm in line filter.

Preferably the agitation is by stirring at about 400-500 rpm, typicallyabout 450 rpm.

Preferably the seed is psilocybin Hydrate A. In one embodiment, 0.1%weight or less of seed is added to the process.

Preferably the crystalline psilocybin is isolated by vacuum filtration.

In one embodiment, the isolated crystals are dried in vacuo at atemperature of at least 30° C., such as between 30 and 50° C., or suchas between 40 and 50° C. In one embodiment, the isolated crystals aredried in vacuo for at least 10 hours, such as between 12 and 18 hours,or such as about 30 hours. In one embodiment, the isolated crystals aredried in vacuo at a temperature of at least 30° C., such as between 30and 50° C., or such as between 40 and 50° C., for at least 10 hours,such as between 12 and 18 hours, or such as about 30 hours. In oneembodiment, the isolated crystals are dried until the isolated crystalslose less than 2% weight in a loss on drying test, such as less than0.5% weight.

Preferably the isolated crystals are washed, several times, in water anddried in vacuo at about 50° C. for at least 12 hours.

The crystals obtained are typically relatively large (range 50 to 200microns) and uniform when viewed under the microscope ×10, asillustrated in FIG. 16 a.

This differs from crystals obtained without controlled cooling which aremuch smaller in size (typically 5 to 50 microns) when viewed under themicroscope ×10, as illustrated in FIG. 16 b.

Stage 1: Synthesis of 1H-indol-4-yl acetate (3)

The core reaction is the reaction of 4-hydroxyindole (1) with aceticanhydride (2) to form 1H-indol-4-yl acetate (3); (FIG. 2)

Most preferably stage 1 is as follows:

4-hydroxyindole (1), DCM (12), and pyridine (13) are added to a vesseland cooled to about 0-5° C. Acetic anhydride (2) is added dropwise, andthe mixture warmed to about 20-25° C. and stirred until complete byHPLC. The reactants are washed with aqueous citric acid solution (14)and aqueous NaHCO₃ (15), dried over MgSO₄ (16) filtered and evaporatedto approximately half volume. Heptane (17) is added, and distillationcontinued to remove the majority of the DCM. The mixture is cooled toabout 5-25° C., filtered, washed with heptane and dried in a vacuum ovenovernight to isolate 1H-indol-4-yl acetate (3) as a solid suitable foruse in the following stage.

Stage 2: Synthesis of3[(dimethylcarbamoyl)carbonyl]-1H-indol-4yl-acetate (6)

The core reaction is the reaction of 1H-indol-4-yl acetate (3) withOxalyl chloride (4) and dimethylamine (5) to form3[(dimethylcarbamoyl)carbonyl]-1H-indol-4yl-acetate (6); (FIG. 3)

Most preferably stage 2 is as follows:

1H-indol-4-yl acetate (3) is dissolved in a mixture of THF (19) and TBME(18) at room temperature. Oxalyl chloride (4) was added dropwiseallowing the reaction to exotherm at about 35-40° C. The temperaturerange is maintained throughout the remainder of the addition. Thereaction is then stirred at about 40° C. until complete by HPLC. Thereaction is cooled to room temperature and heptane (17) added resultingin precipitation of further solids. The slurry is stirred, then allowedto settle, followed by removal of the majority of the solvent (18/19) bydecanting. The solid was washed in the vessel twice with heptane (17).TBME (18) is added to give a yellow slurry and the mixture cooled toabout −20° C. Dimethylamine solution (5) is added maintaining thetemperature at −20° C. to −10° C. The reaction was then warmed to roomtemperature and stirred until complete, adding extra dimethylamine ifnecessary. The reaction was filtered, washed with heptane (17) and driedin a vacuum oven. The crude3[(dimethylcarbamoyl)carbonyl]-1H-indol-4yl-acetate (6) was furtherpurified by a slurry in water (20), then IPA (21) and then dried in avacuum oven to yield (6) as a solid suitable for use in the followingstage.

Stage 3: Synthesis of 3-(2-(dimethylamino) ethyl)-1H-indol-4-ol(Psilocin) (8)

The core reaction is the reaction of3[(dimethylcarbamoyl)carbonyl]-1H-indol-4yl-acetate (6) with lithiumaluminium hydride (7) to form psilocin (8); (FIG. 4)

Most preferably stage 3 is as follows:

The 3[(dimethylcarbamoyl)carbonyl]-1H-indol-4yl-acetate (6) was slurriedin THF (19) and cooled to about 0° C. A THF solution of LiAlH₄ (7) wasadded dropwise maintaining the temperature at about 0-20° C. Thereaction was then refluxed until complete by HPLC. The reaction wascooled to 0° C. and the excess LiAlH₄ quenched by addition of acetone(22) followed by aqueous citric acid solution (14). The batch wasfiltered to remove Lithium and Aluminium salts. The filtrate was driedover MgSO₄ (16), filtered and concentrated and loaded onto a silica pad(23). The pad was eluted with THF (19) and the product containingfractions evaporated. The resulting solid was slurried in iPrOAc:TBME(24/18) mixture, filtered and washed with TBME. The solid was dried inthe oven to yield high purity psilocin (8) as an off white solid.

Stage 4: Synthesis of benzyl3-[2-(benzyldimethylazaniumyl)ethyl]-1H-indol-4-yl phosphate (10)

The core reaction is the reaction of psilocin (8) withtetrabenzylpyrophosphate (9) to form benzyl3-[2-(benzyldimethylazaniumyl)ethyl]-1H-indol-4-yl phosphate (10), (FIG.5)

Most preferably stage 4 is as follows:

Charge psilocin (8) to a vessel followed by THF (19). The reaction wascooled to −50° C. to −70° C. and NaHMDS (25) was added dropwise at about−45° C. to −70° C. The temperature was adjusted to about −45° C. to −60°C. and tetrabenzylpyrophosphate in THF was added. The batch was allowedto warm to 0° C. after which the solid by products were removed byfiltration and the filtrate concentrated in vacuo. The concentratedmixture was then heated to about 40° C. and stirred until theintermediate had converted to the stage 4 product (10)—controlled bymonitoring and the use of HPLC. The batch was cooled to about 0-5° C.and the resulting solid isolated by filtration and dried in vacuo toprovide benzyl 3-[2-(benzyidimethylazaniumyl)ethyl]-1H-indol-4-ylphosphate (10) as a solid.

Stage 5: Synthesis of Intermediate Grade 3-[2-(dimethylazaniumyl)ethyl]-1H-indol-4-yl hydrogen phosphate (Psilocybin Crude) (12)

The core reaction comprises reacting benzyl3-[2-(benzyidimethylazaniumyl) ethyl]-1H-indol-4-yl phosphate (10) withhydrogen (11) to form psilocybin (12), (FIG. 6).

Most preferably stage 5 is as follows:

To a vessel was charged Pd/C (26), methanol (24) and3-[2-(benzyidimethylazaniumyl)ethyl]-1H-indol-4-yl phosphate (10) andthe resulting mixture sparged with hydrogen (11) until complete by HPLC.Purified water (20) is added during this process to retain the productin solution. The mixture was heated to about 35° C.-45° C. and thenfiltered through a bed of Celite (27) washing with methanol (24) andpurified water (20). The filtrate was evaporated in vacuo, azeotropingwith ethanol (28) to obtain intermediate grade psilocybin (12).

Stage 6: Synthesis of 3-[2-(dimethylazaniumyl) ethyl]-1H-indol-4-ylhydrogen phosphate (Psilocybin)

The core purifying/polymorph determining step is a water crystallizationstep, followed by a controlled cooling and drying step, to produce highpurity crystalline psilocybin, Polymorph A or Polymorph A′.

Most preferably stage 6 is as follows:

The intermediate grade Psilocybin (12) (stage 5) was charged to a vesselwith purified water (20) and the mixture heated until the psilocybin(12) dissolved. The resulting bulk solution was then polish filteredinto a pre-warmed vessel. The temperature was adjusted to, preferably,about 68° C.-70° C., and a Psilocybin hydrate seed (i.e., Hydrate A) wasadded to the reaction. The batch was then cooled in a controlled mannerto about 0-10° C. and stirred, and the solids were collected byfiltration and washed with purified water. The isolated solids were thendried in vacuo to yield high purity crystalline Psilocybin, Polymorph Aor A′, as an off white solid.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention are further described hereinafter withreference to the accompanying drawings, in which:

FIG. 1 is a schematic of the reaction taught in JNP;

FIG. 2 is a schematic of the Stage 1 reaction of one aspect of thepresent invention;

FIG. 3 is a schematic of the Stage 2 reaction of one aspect of thepresent invention;

FIG. 4 is a schematic of the Stage 3 reaction of one aspect of thepresent invention;

FIG. 5 is a schematic of the Stage 4 reaction of one aspect of thepresent invention;

FIG. 6 is a schematic of the Stage 5 reaction of one aspect of thepresent invention;

FIG. 7a is a XRPD diffractogram of Polymorph A (GM764B);

FIG. 7b is a XRPD diffractogram of Polymorph A′ (JCCA2160F);

FIG. 7c is a XRPD diffractogram of Polymorph B; (JCCA2160-F-TM2);

FIG. 7d is a XRPD diffractogram of a Hydrate A (JCCA2157E);

FIG. 7e is a XRPD diffractogram of an ethanol solvate (JCCA2158D);

FIG. 7f is a XRPD diffractogram of product obtained during developmentof the process (CB646-E) (top)—compared to the diffractograms PolymorphA′ (JCCA2160F) (middle) and Polymorph B (JCCA2160-TM2) (bottom);

FIG. 8a is a DSC and TGA thermograph of Polymorph A (GM764B);

FIG. 8b is a DSC and TGA thermograph of Polymorph A′ (JCCA2160F);

FIG. 8c is a DSC thermograph of Polymorph B (GM748A);

FIG. 8d is a DSC and TGA thermograph of Hydrate A (JCCA2157E);

FIG. 8e is a DSC and TGA thermograph of ethanol solvate (JCCA2158D);

FIG. 9 is a form phase diagram showing the inter-relationship of form inwater-based systems;

FIG. 10 is a 1H NMR spectrum of Psilocybin; (Read alongside assignmentExample 7);

FIG. 11 is a ¹³C NMR spectrum of Psilocybin; (Read alongside assignmentExample 7);

FIG. 12 is a FT-IR Spectrum of Psilocybin;

FIG. 13 is a Mass Spectrum of Psilocybin;

FIG. 14 is a numbered structural formula of Psilocybin;

FIG. 15 is a temperature solubility curve for Psilocybin in water;

FIG. 16a is a micrograph showing crystals obtained by controlledcooling;

FIG. 16b is a micrograph showing crystals obtained by uncontrolledcooling drying;

FIG. 17 is a form phase diagram showing the inter-relationship of formsin different solvent systems;

FIG. 18 is XRPD diffractogram—Pattern C for solids isolated at 25 and50° C.;

FIG. 19 is XRPD diffractograms—Patterns D, E and F for solids isolatedat 25 and 50° C.;

FIG. 20 is a comparison of the XRPD diffractograms acquired for thesolids isolated from the equilibration of amorphous Psilocybin insolvents A to H;

FIG. 21 is a comparison of the XRPD diffractograms acquired for thesolids isolated from the equilibration of amorphous Psilocybin insolvents I to P; and

FIG. 22 is a comparison of the XRPD diffractograms acquired for thesolids isolated from the equilibration of amorphous Psilocybin insolvents R to Y.

DETAILED DESCRIPTION

In contrast to the prior art, the present invention sought to producepsilocybin at a commercial large scale, in amounts or batches of atleast 100 g, and more preferably at least 250 g, levels 1 log or 2 logshigher than the levels described in JNP, which describes a “large” scalemethod to producing gram quantities on a 10 g scale.

To demonstrate the many significant development steps from JNP, thedescription below sets out details of experiments and investigationsundertaken at each of the process stages, which illustrate theselections made to overcome the numerous technical problems faced, inproducing psilocybin (7) to GMP at a large scale (including the variousintermediates (2-6)) starting from 4-hydroxyindole (1).

Reference to a particular numerical value includes at least thatparticular value, unless the context clearly dictates otherwise. When arange of values is expressed, another embodiment includes from the oneparticular value and/or to the other particular value.

Further, reference to values stated in ranges include each and everyvalue within that range.

All ranges are inclusive and combinable.

When values are expressed as approximations, by use of the antecedent“about,” it will be understood that the particular value forms anotherembodiment.

As used herein, the singular forms “a,” “an,” and “the” include theplural.

The term “about” when used in reference to numerical ranges, cut-offs,or specific values is used to indicate that the recited values may varyby up to as much as 10% from the listed value. As many of the numericalvalues used herein are experimentally determined, it should beunderstood by those skilled in the art that such determinations can, andoften times will, vary among different experiments. The values usedherein should not be considered unduly limiting by virtue of thisinherent variation. Thus, the term “about” is used to encompassvariations of ±10% or less, variations of ±5% or less, variations of ±1%or less, variations of ±0.5% or less, or variations of ±0.1% or lessfrom the specified value.

As used herein, “treating” and like terms refer to reducing the severityand/or frequency of symptoms, eliminating symptoms and/or the underlyingcause of said symptoms, reducing the frequency or likelihood of symptomsand/or their underlying cause, delaying, preventing and/or slowing theprogression of diseases and/or disorders and improving or remediatingdamage caused, directly or indirectly, by the diseases and/or disorders.

The following abbreviations have been used herein:

-   DSC—Differential Scanning Calorimetry-   RT—room temperature-   TBME—methyl tert-butyl ether-   TGA—Thermogravimetric Analysis-   THF—tetrahydrofuran-   wrt—with respect to-   XRPD—X-Ray Powder Diffraction

Example 1

Stage 6 Crystalisation Process and Resulting Polymorphs

Experimental to Produce Form A′:

1.0 g of crude Psilocybin was charged to a 25 mL flask. Water (12.8mL/16 volumes based on activity of input material) was added. Themixture was agitated and heated to 80° C. A dark brown solution withvisible undissolved solids was obtained. The mixture was polish filteredthrough a warmed 0.45 μm filter into a hot 25 mL flask. The undissolvedsolids were removed to give a dark brown solution. The solution wasre-equilibrated at 75° C. and then cooled slowly (10° C./hour) toambient temperature. The resulting pale brown solution was equilibratedat ambient temperature for 16 hours. The suspension was cooled to 5° C.prior to isolation of the solid by vacuum filtration. The filter cakewas washed with water (0.8 mL/1 volume) and dried in vacuo at 50° C. for16 hours. Yield of 75%, chemical purity 99%, NMR assay>98%.

The procedure above was repeated with 14 volumes (11.2 mL) of water.Yield of 69%, chemical purity 99%, NMR assay>98%.

In both cases, dissolution of crude Psilocybin was achieved at ca. 75°C. On gradual cooling, precipitation was observed at ca. 60° C.

In both cases, psilocybin Polymorph A′ was produced, confirmed by XRPD(diffractogram consistent with FIG. 7b ) and DSC (thermogram consistentwith FIG. 8b ).

Experimental to Produce Form A:

94 g of crude Psilocybin obtained from the Stage 5 process (about 93%pure by HPLC with about 4% pyrophosphate impurity) was subject to anaqueous re-crystallisation as set out below:

The protocol used sufficient water (12 volumes), a rapid agitation rate(450 rpm) and a controlled cooling profile (10° C./hr).

Psilocybin (94.0 g) (CB650E) was charged to a 2 L flask. Water (902 ml,12 volumes based upon activity of input material) was added. The mixturewas agitated and heated to about 78° C. A dark brown solution withvisible undissolved solids was obtained. The mixture was polish filteredthrough a 1.21 μm in-line filter into a hot 5 L flask fitted with anoverhead stirrer (450 rpm). The undissolved solids were removed to givea clarified dark brown solution. The solution was re-equilibrated atabout 75° C. for 15 minutes and then cooled slowly (10° C./hour) toambient temperature. The solution was seeded with Psilocybin Hydrate A(GM758A-XRPD diffractogram consistent with FIG. 7d ) followingmaturation in water) at 68° C.-70° C. The resulting pale brownsuspension was equilibrated at ambient temperature for about 16 hours.The suspension was cooled to 5° C. for one hour prior to isolation ofthe solid by vacuum filtration. The filter cake was washed with water(282 mL, 3 volumes) and dried in vacuo at about 50° C. for 30 hours.

The process was completed successfully with a yield of 75% achieved.Chemical purity of the solid was confirmed as 99.3%. Analysis of thesolid by XRPD post drying for 30 hours showed Polymorph A (FIG. 7a ). Acharacteristic perturbation was observed at ca ˜17 °2θ, such as 17.5°2θ, and was pronounced in the bulk material.

Solid State Characterisation of Polymorph A and Polymorph A′

The DSC and TGA thermograms (FIG. 8a ) obtained for Polymorph A werecomparable to the DSC and TGA thermograms obtained for Polymorph A′(FIG. 8b ). The TGA thermograms (FIG. 8a ) obtained for Polymorph A andPolymorph A′ show no weight loss prior to decomposition. This suggestedthat the difference between the XRPDs obtained for Polymorph A (FIG. 7a; perturbation present at ca. ˜17 °2θ) and for Polymorph A′ which wasobtained at small scale (FIG. 7b ; perturbation not present) was not dueto excess hydration.

Microscopy of the solid (FIG. 16a ) shows rod shaped crystals with gooduniformity with a size range of between 50 and 200 micron.

The XRPD diffractogram obtained for Polymorph A′ does not demonstrate aperturbation at ca. ˜17 °2θ to the same extent as Polymorph A. Theperturbation in the XRPD diffractogram at ca. ˜17 °2θ is more pronouncedfor psilocybin produced at large scale (compared to that obtained atsmall scale) and was unexpected. Applicant has demonstrated that theHydrate A is the only polymorphic form that exists across a range oftemperatures with no diffraction peak in the 17 °2 theta region (seeFIG. 7d . This strongly suggests a collapse of Hydrate A upondehydration to yield Polymorph A or A′ that varies with scale and thatPolymorph A is the true form with Polymorph A′ formed at a small scalebeing atypical.

To test the robustness of this theory and to demonstrate a return toPolymorph A, a small portion of the bulk was re-dried following anothersoak in water (to reproduce Hydrate A). A small sample (250mg—psilocybin Polymorph A) was equilibrated in water (10 vols) for onehour. The suspension was filtered and analysis of the damp solidconfirmed that Hydrate A had been generated (FIG. 7d ), no perturbationat 17 °2 theta. The material was dried in vacuo for 16 hours and thesolid reassessed by XRPD. Polymorph A′ material was confirmed by XRPD(FIG. 7b ) with the reduction in the XRPD perturbation noted. Additionaldrying of the original bulk solid and ageing at ambient temperature didnot change the XRPD diffractogram of the solid. The two solid versionsobtained, the XRPD diffractograms for Polymorph A and Polymorph A′ arevirtually identical other than the ˜17.5° 2 theta peak. The thermalproperties are also identical. The distinction between the XRPDdiffractograms for Polymorph A versus Polymorph A′ is subtle and bothpolymorphs kinetically convert to the hydrated state very rapidly.

Additional experiments were performed to ascertain if the differences inthe XRPD diffractograms for Polymorph A and Polymorph A′ were due to thelarger scale crystallisation process delivering solids of a largerparticle size that subsequently did not dry as effectively and causedthe change, or whether the habit and size difference of the crystallinesolid was the cause. Psilocybin Polymorph A (polymorphic form confirmedprior to experiment) was ground via a mortar and pestle and assessed byXRPD. No change in polymorph was observed. Another portion (51 mg) wascharged with water (<1 mL) and assessed damp to confirm that the hydratewas formed. Both lots were dried in vacuo at 50° C. for ca. 18 hours andre-assessed by XRPD. The ground sample remained as Polymorph A. Thehydrated sample after dehydration was shown to be Polymorph A′ (i.e., noreflection at ˜17.5 °2θ). This suggested that size/habit alone were notthe sole reason for the original reflection peaks.

TGA assessment revealed that the input lot demonstrated a small massloss (0.139% weight) by ca. 70° C. The particle size reduced andsubsequently dried solid demonstrated a greater mass loss of 0.343 wt %by ca. 75° C. whereas the hydrated and dried solid demonstrated thesmallest mass loss of 0.069 wt %/o by ca. 80° C. The particle sizereduced and subsequently dried solid was held at 80° C. for 10 minutes(past the point of mass loss by TGA) but assessment by XRPD revealed nochange from the input, meaning that low levels of hydration and partialswelling of the crystalline lattice were not the cause of the variation

It is possible to generate Polymorph A′ via the hydration of Polymorph Aand subsequent drying of the isolated solid on a small scale.

Psilocybin Polymorph A and Polymorph A′, ca. 60 mg each, were chargedwith water, 0.2 ml, to deliver Hydrate A from both lots. Half of eachHydrate A was dried in vacuo at 25° C. for ca. 17¼ hours and theremainder of each Hydrate A was dried at ambient temperature under astream of N₂ for Ca. 171/4 hours. The solids were isolated followingdrying and assessed by XRPD. XRPD assessment of the solids isolated fromthe Polymorph A input confirmed that Hydrate A was successfullygenerated and that the solids dried to give Polymorph A′ from bothdrying methods. XRPD assessment of the solids isolated from thePolymorph A′ input confirmed that Hydrate A was successfully generatedand that the solids dried to remain as Polymorph A′ from both dryingmethods.

On the small scale investigated, Polymorph A and Polymorph A′ will dryto give Polymorph A′ via conversion to Hydrate A.

Psilocybin Polymorph A (100 mg) was particle size reduced via mortar andpestle grinding. The ground lot was subject to two different dryingregimes in order to assess whether reducing the particle size affectedthe dehydration of the sample. The first sample was held at 80° C. for10 minutes and the second sample held at 110° C. for 10 minutes. Bothsolids were assessed by XRPD which revealed that Polymorph A wasretained. It was considered whether the ground lot in the priorisothermal stresses were not held at 110° C. for long enough to impactthe form and so a portion of the ground lot was dried in vacuo at 110°C. for ca. 24 hours. Assessment by XRPD revealed a subtle change in formwith the Polymorph A reflections at ca. 17 still present but at aslightly reduced intensity.

It was concluded that Polymorph A would not readily convert to PolymorphA′ via particle size reduction and/or drying at high temperature.

Methodology

Stability assessments of Psilocybin, not containing the pyrophosphateimpurity, indicated that at temperatures in excess of 80° C. the levelof the Stage 3 intermediate impurity (psilocin) generated by hydrolysisof Psilocybin is of concern. For example, when a 83 mg/mL Psilocybinaqueous solution is heated to 90° C. and analysed by HPLC at 1, 2 and 4hours, the level of the Stage 3 impurity were determined as 0.28, 1.82and 7.79 area % respectively. In comparison, when 50 mg of psilocybin isdissolved in water (1.2-1.8 ml; volume sufficient to maintain asolution) and heated to 70, 75 and 80° C. for 4 hours, the level of theStage 3 impurity was determined, by HPLC, as 0.53, 0.74 and 2.30 area %respectively. The recrystallization heats the crude Psilocybin tobetween 75° C. and 80° C. in order to achieve dissolution, and polishfiltration. The immediate cooling of the solution limits the level ofPsilocybin hydrolysis by reducing the residency time of the material toexcessive temperature.

Further trial re-crystallisation's of Psilocybin were conductedintroducing the following variations:

Varying the volumes of water used;

Varying agitation;

Having a controlled cooling profile;

Having a rapid (uncontrolled) cooling profile.

Using smaller volumes of water (as little as 12 volumes) did not hinderthe re-crystallisation process and dissolution of Psilocybin wasachieved at a temperature to enable a polish filtration step. Differentcooling rates were shown to result in different crystal sizedistributions; a slow controlled cool at ca 10° C. per hour produced arelatively larger and more even average crystal size (FIG. 16a ) whereasa rapid cooling profile delivered smaller crystals (FIG. 16b ). Acontrolled cooling profile is preferred and this was reflected in animproved purity for the controlled cool.

Using the process resulted in Psilocybin of 99.3% chemical purity, witha 75% yield. Thermal characteristics of the solid corresponded withthose desired. Differences in the XRPD diffractogram of the dry solidhave suggested that the drying profile may be important in determininghow the Hydrate A collapses to give the preferred solid form. PolymorphA has been demonstrated to be stable under accelerated stability testingconditions for 12 months.

Experimental

Stage 5 was charged to vessel under N₂ followed by water (approx. 12-15vol based on active Stage 5). The mixture was heated to about 80° C. toachieve dissolution and polish filtered through a 1.21 μm in-line filterinto a clean new flask heated to 80° C. The agitation rate was set to ahigh level (450 rpm) and the solution equilibrated at 70-75° C. Thesolution was cooled to ambient temperature, at approx. 10° C./hour,seeding with Psilocybin Hydrate A (0.001×stage 5 charge) at 68-70° C.The suspension was held at ambient temperature overnight then cooled toapprox. 5° C. and held for 1 hour. The suspension was filtered, washingwith water (2-3 volumes based upon active charge of Stage 5). The purePsilocybin was dried in vacuo at 50° C. Crystalline material psilocybin(Polymorph A or Polymorph A′ dependent on scale) was obtained, forexample using 94 g input of psilocybin yielded Polymorph A and using 1 ginput of psilocybin yielded Polymorph A′. Typically, batch sizes ofgreater than 5 g deliver Polymorph A, while batch sizes less than 5 gdeliver Polymorph A′.

The differences from JNP and the benefits can be summarised as follows:

i) This additional crystallisation step gives rise to a definedcrystalline form—Polymorph A (or A′).

ii) Heating to about 80° C., for a short period, has the advantage thatsolubility is maximised (and hydrolysis avoided), which ensures goodyields.

iii) At about 70-80° C. polish filtration can be used to removeinsoluble impurities. This is best achieved using an in-linefilter—typically about 1.2 μm. This ensures good chemical purity.

iv) Using a high agitation rate (typically about 450 rpm) ensures speedydissolution allowing the time at which the solution is kept at 80° C. tobe minimised, thus avoiding increased levels of the Stage 3 intermediateimpurity formed by hydrolysis of Psilocybin.v) The provision of controlled cooling, typically cooling at about 10°C. per hour, delivers a more uniform crystal size and maintains form ascrystalline Hydrate A.vi) Seeding the solution at about 70° C. with Psilocybin Hydrate Afacilitates crystallisation as the Hydrate A.vii) The crystals are washed in water and dried at about 50° C. tomaximise purity and deliver Polymorph A or A′ depending on scale.

Examples 2 to 6

Stages 1 to 5 Production of Psilocybin

The following Examples illustrate significant developments from theprocess described in JNP, and illustrated in FIG. 1, as described hereinbefore.

Example 2

Stage 1 (FIG. 2)

The stage 1 conditions in JNP used 1.1 eq Ac₂O, and 1.2 eq Pyridine inthe solvent DCM. The reaction was found to be complete (99% product, 0%SM) after stirring overnight. The reaction mixture was washed with waterand concentrated in vacuo giving a brown oil. In the literature, the oilwas taken up in EtOAc and concentrated by evaporation, givingprecipitation of solids at low volume.

Investigation

However, in the Applicants hands precipitation of solids from EtOAc wasnot observed. Precipitation of solids was encouraged by trituration withheptane, however this would not form a scalable process. The solids werecollected giving high purity stage 1 product (75% yield, >95% pure byNMR).

While the reaction worked well, the isolation procedure required furtherdevelopment in order that an easy to handle solid could be obtained. Itwas hoped that isolation of the solids by filtration would then alsooffer a means of purification.

The reaction was first trialed in EtOAc to see if precipitation ofsolids could be encouraged allowing isolation directly from the reactionmixture. However, the reaction profile in EtOAc was found to be lessfavourable than in DCM and therefore the reaction was abandoned.

Applicant washed out the pyridine from the DCM reaction mixture, as itwas believed this may be preventing re-crystallisation of the product.The reaction was repeated (completion of 0.4% SM, 98.7% product by HPLC)and the reaction mixture washed with 20% citric acid to achieve pH 2/3,removing pyridine and then saturated NaHCO₃ (aq) to avoid low pH in theevaporation steps. The organics were dried and a solvent swap to heptanewas carried out giving precipitation of stage 1. The solids werecollected by filtration yielding pure stage 1 after drying in vacuo (87%yield, >95% purity by NMR).

Stability trials were carried out that confirmed the reaction mixturewas stable overnight when stirred with 20% citric acid, and alsosaturated NaHCO₃. The product was found to be stable when oven dried at40° C. and 60° C.

Scale Up

The stage 1 reaction was successfully scaled up processing >100 g of4-hydroxyindole. The reaction progressed as expected and was worked upto give stage 1 product (93% yield, ˜98% NMR purity).

GMP Raw Material Synthesis

A large scale stage 1 reaction was carried out to supply GMP startingmaterial (processing >500 g of 4-hydroxyindole). The reaction proceededas expected giving consumption of SM by HPLC (99.2% product, <0.1% SM).The reaction was worked up using the established procedure to give stage1 product after drying (94% yield, 99.1% by HPLC, 99% NMR assay).

It was also noted in development that the stage 1 procedure waseffective in removing minor impurities present in some batches of4-hydroxyindole. The low level impurities present in 4-hydroxyindolewere completely removed after the stage 1 reaction providing cleanmaterial in high yield (89%) and purity (99% by HPLC, 99% by NMR assay).

Experimental

4-hydroxyindole (1 eq. limiting reagent) was charged to a vessel underN₂ followed by DCM (dichloromethane; 6 vol based on 4-hydroxyindolecharge). The reaction was cooled to 0-5° C. and pyridine added (1.2 eq)dropwise at 0-5° C. Acetic anhydride (1.1 eq) was added dropwise at 0-5°C. and the reaction warmed to 20-25° C. for 1-1.5 hrs and stirred at20-25° C. for a further 3 hours. The reaction was sampled and analysedfor completion. The reaction was then washed three times with 20%aqueous citric acid solution (3×3 vol based on 4-hydroxyindole charge)and once with sat. NaHCO₃ (3 vol based on 4-hydroxyindole charge). TheDCM solution was dried over MgSO₄ and filtered and the DCM layerconcentrated to half volume by distillation. Heptane (6 vol based on4-hydroxyindole charge) was added and further DCM was removed bydistillation until full precipitation of the Stage 1 had occurred. Thereaction was cooled to 15-25° C. and the solids collected by filtration,washing with heptane (1 vol based on 4-hydroxyindole charge) dried undervacuum overnight at 60° C.

The differences from JNP and the benefits can be summarised as follows:

i) Applicant washed out the pyridine using citric acid at a pH of about2-3. This facilitated improved isolation and crystallisation. Inpractice the DCM phase is separated and the aqueous citric acid phasediscarded.

ii) An additional wash in sodium bicarbonate resulted in furtherimprovement.

iii) A solvent swap to heptane improved solid precipitation maximisingyield and resulting in reproducible high purity Stage 1.

Example 3

Stage 2 (FIG. 3)

Step i—Acid Chloride Formation

Formation of reactive Intermediate 2A by reaction of the stage 1 productwith oxalyl chloride (1.5 eq) was initially trialed in a mixture of TBMEand THF (6 vol/1 vol) to determine if it was a viable alternative tovolatile and highly flammable Et₂O as used in the literature. Thereaction gave completion after ˜18 hours with a similar solubilityprofile to Et₂O (stage 1 in solution, precipitation of stage 2A).

As the acid chloride intermediate is prone to hydrolysis, leading tovariable analytical results a more robust sample make up and analysiswas developed in which the reaction was quenched into THF/NMe₂ (to givestage 2) and then analysed by HPLC.

The ratio of TBME and THF was optimised to give the highest purity andyield of intermediate with a preferred ratio of TBME:THF of 6:1 chosenfor scale up. Other ratios of TBME:THF may be used.

A scale up reaction was carried out using the preferred solvent mixture(1 vol THF, 6 vol TBME) but with the oxalyl chloride addition carriedout at 30-35° C. The resulting solution was then heated at 40° C. for2.5 hrs giving a completion with ˜1% stage 1 product remaining. Carryingout the addition hot to maintain a solution ensures a high reaction rateand gave an improved level of completion with a much shorter reactiontime (2.5 hrs vs overnight). The product was still seen to precipitateat temperature after ˜15 min and no detrimental effect on the reactionprofile was observed by HPLC.

As the stability of Intermediate 2A was not known, telescoping ofmaterial through to stage 2 was attempted rather than isolating theintermediate and risking degradation (hydrolysis). The reaction profilewas complex with multiple components present at a low level. TBME wasadded and the precipitate collected. However, this was also found to bea complex mixture by HPLC/NMR.

Due to the poor reaction profile it was deemed necessary to isolateIntermediate 2A to allow for purification away from excess oxalylchloride. The reaction was repeated and the yellow precipitate collectedby filtration and washed with TBME to remove the excess oxalyl chloride(80% yield). NMR analysis confirmed the product to be of sufficientpurity (˜95% by NMR). However despite storing under nitrogen, somedecomposition was noted over the following days giving partialhydrolysis including de-protection of the acetate group.

In order to try and reduce the potential for hydrolysis of theintermediate acid chloride during isolation, further investigations intoa telescoped procedure were carried out. It was found that by allowingthe reaction mixture to settle the TBME liquors could easily be decantedand then the residual solids washed with further portions of TBME in asimilar manner. This allowed purification of Intermediate 2A away fromexcess oxalyl chloride whilst minimising exposure to moisture.

It was felt that some reaction yield may be lost due to partialsolubility of the intermediate in the THF/TBME mixture. This wasconfirmed by adding heptane to the decantation liquors which gaveprecipitation of further solids. To limit this solubility, a heptane (8vol) addition was made prior to decantation. Rather than washing thesolids with TBME, heptane was also used for the washes (3×6 vol) whichmaximised the yield while maintaining the high purity of theintermediate. This methodology was successfully scaled up and is thepreferred process.

Step ii—Reaction with Dimethylamine

The literature (Synthesis, 1999, 6, 935-938; D. E. Nichols) suggestedHNMe₂ gas was effective for this transformation. However to simplifylarge scale processing this was substituted for either solid HNMe₂.HCl,with an additional excess of base, or a solution of HNMe₂ in THF. JNPuses HNMe₂ in the presence of excess base (pyridine).

Initially isolated Intermediate 2A was used to optimise the reactionwith dimethylamine via a series of trial reactions (see Table 11).

TABLE 11 Stage 2 reaction optimisation trials Dimethylamine HPLC #Source Base Solvent completion Isolated solids 1 2M HNMe₂/ PyridineTHF/TBME 80% 64% yield, ~70% THF 1.2 eq 1.3 eq product, pure by NMR, 2HNMe₂•HCl K₂CO₃ THF/H₂O 70% 40% yield ~98% 1.5 eq product by NMR. 3 2MHNMe₂/ Pyridine Et₂O 81% 63% yield, ~90% THF 1.33 eq 3.6 eq product byNMR. 4 2M HNMe₂/ N/A Et₂O 93% 72% yield, ~98% THF 2.9 eq product by NMR.

The literature supplied conditions with pyridine (#1) were trialed alongwith a similar reaction in Et₂O (#3), a biphasic reaction usingMe₂NH.HCl and aqueous K₂CO₃ (#2) and a reaction with excess 2M Me₂NH inTHF (#4). The major component by HPLC was the desired product in allcases with the conditions using aqueous base and excess Me₂NH beinggenerally much cleaner than those with pyridine. While significanthydrolysis product was seen in all cases this was thought to be theresult of unreacted Intermediate 2A which was quenched during samplemakeup for HPLC analysis. The reactions were worked up by addition ofwater and then the organic solvent was removed in vacuo givingprecipitation of solids.

The reaction with excess amine showed a much improved impurity profilewhich translated into a higher yield (72% vs 63%) and purity (98% vs90%). This approach limited the water content of the reaction andtherefore minimised the opportunity for hydrolysis to occur.Purification was also expected to be more facile due to the absence ofpyridine in the isolated solids. For these reasons the conditions withexcess HNMe₂ as base were chosen for scale up.

The reaction in Et₂O gave a clean (#4) profile. However, to facilitatelarge scale processing it proved advantageous to switch to a lessvolatile solvent, such as TBME. This would facilitate telescoping theacid chloride into this reaction. For these reasons it was chosen tocarry out the reaction in TBME using excess 2M Me₂NH in THF.

It was believed that the addition of water would aid the workup bysolubilising the HNMe₂.HCl salts that were present and resulted in avery thick mixture and slow filtrations. This was trialed. However, whenwater was added to the reactions in TBME and THF a poor recovery wasobtained with analysis of the liquors showing additional impurities andextensive acetate de-protection (phenol product). Further development ofthe purification was therefore required.

Purification Development

It was desirable to develop a purification strategy that would removethe hydrolysis product and other impurities observed. It was alsodesirable to include water in the crystallisation to reduce the saltcontent of the isolated material (assumed HNMe₂.HCl). To this end aseries of 15 solvents and solvent mixtures were screened (100 mg scale,10 vol solvent, heat cycling to 60° C.).

TABLE 12 Stage 2 purification trials HPLC Purity (hydrolysis imp ofSolvent Observations Recovery Intermediate 2A) TBME Slurry 47 mg* 96.4%(2.8%) DCM Slurry 60 mg 99.5% (0.5%) Toluene Slurry 30 mg* 95.8% (3.4%)EtOAc Slurry 66 mg* 97.8% (1.6%) iPrOAc Slurry 40 mg* 97.3% (2.0%) IPASlurry 65 mg 99.4% (0.4%) EtOH/H₂O, 1:1 Slurry 62 mg 99.4% (0.5%)MeCN/H₂O, 1:1 Partial solution 30 mg 99.0% (0.7%) at RT Solution at 60°C. Acetone/H₂O, 1:1 Slurry at RT 51 mg 99.4% (0.5%) Solution at 60° C.THF/H₂O, 1:1 Partial solution No precipitate n/a at RT Solution at 60°C. Heptane Slurry 34 mg* 95.0% (3.6%) MIBK Slurry 65 mg 97.9% (1.4%) MEKSlurry 60 mg 99.6% (0.3%) Cyclohexane Slurry 41 mg* 94.4% (4%) XylenesSlurry 56 mg* 96.1% (3%) *Recovery not representative due to thicksuspensions and solids adhering to the glass vial.

From the solvents screened acetone/water gave a re-crystallisation withlittle observed solubility at room temperature. Since this was anaqueous system it had the advantage of helping to purge Me₂NH.HCl fromthe solids.

The acetone/water re-crystallisation was scaled up. A solution wasobtained at temperature (5 vol acetone, 1 vol water) prior to additionof further water (4 vol) and the mixture cooled to RT givingcrystallisation (62% recovery, >99% HPLC purity). This process wassubsequently scaled up further with addition of more water to aid therecovery (in total 5 vol acetone, 10 vol water, 78% recovery).

The process was scaled up further (30 g) and the crude solids takenthrough the re-crystallisation procedure. While product purity was high,there was a drop in yield (56% yield, 99% by NMR assay, 99.4% by HPLC).

In order to improve the recovery, the amount of water added was furtherincreased from 10 vol to 15 vol. This maintained product purity atgreater than 99% and gave a higher recovery on a small scale (90%recovery, 56-70% previously observed). However, scale up of this amendedprocedure again gave a low recovery (58% yield). Therefore, due to theissues encountered when scaling up the re-crystallisation, analternative means of purification was sought based on the originalslurry screen that was carried out (Table 12 above).

Redevelopment of the purification strategy took place using materialisolated from a large scale, stage 2, reaction. The reaction progressedas expected to give crude product after oven drying (70% by NMR assay,79% active yield). To remove the significant salt component (presumed tobe HNMe₂.HCl) a portion was water slurried at RT. After drying this gave75% recovery (95% by NMR assay) showing this to be an effective means ofreducing the salt content. HPLC purity remained unchanged at ˜93%. Amethod was then sought to increase the chemical purity of the solids.

From the initial screen both EtOH:H₂O, IPA and acetone:H₂O appeared togive high purity product with a good recovery and so these solventsystems were chosen for further investigation. Input purity was 92.7%with the main impurities at levels of 1.4%, 1.0% and 0.8%.

TABLE 13 Further purification development (slurry at 40° C. 1 hr,filtration at RT, 1 vol wash) HPLC purity (main impurity levels) Solventsystem Recovery Product Imp 1 Imp 2 Imp 3 Acetone/Water 67% 98.8% 0.7%0.2% 0.1% 6/16 vol 1:1 EtOH:H₂O 79% 98.0% 0.9% 0.5% 0.2% 10 vol IPA 90%97.5% 0.8% 0.8% 0.3% 10 vol IPA 92% 97.2% 1.0% 0.7% 0.3% 5 vol 4.5:2.5IPA:H₂O * 79% 98.7% 0.6% 0.4% 0.1% 7 vol 6:1 IPA:H₂O   82% ** 98.0% 0.9%0.6% 0.2% 7 vol * The reaction was initially run in 1:1 IPA:H₂O at 5vol. However it became too thick to stir and so a further 2 vol of IPAwas added. ** The mixture was thick and the solids present were veryfine making filtration difficult with some solids beating the filter.

The results of these trials suggested that good recoveries were possiblefrom these systems, particularly those based on IPA. EtOH:H₂O gave amarginally better impurity profile than IPA alone; however the recoverywas not as good (79 vs 90%). The impurity profile with IPA was greatlyenhanced by the presence of water (98.7% vs ˜97.5%) however this led toa lower recovery (79 vs 90%). This suggested a certain level of watersolubility for the compound. A final trial in IPA and EtOH:Water wasconducted with reduced water volumes to see if a balance could be foundthat provided high purity and a recovery of ˜90%. While this systemimproved the yield the filtration was slow and therefore further solventmixtures were also evaluated.

TABLE 14 Examination of further solvent mixtures (500 mg scale, slurryat 40° C. 1 hr, filtration at RT, 1 vol wash) HPLC purity (impuritylevels) Solvent system Recovery Product Imp 1 Imp 2 Imp 3 IPA 460 mg(92%) 97.6% 1.1% 0.7% 0.3% 10 vol 1:3 EtOH:H2O 451 mg (90%) 96.2% 1.4%0.8% 0.5% 5 vol 1:2 EtOH:H2O 440 mg (88%) 97.5% 1.3% 0.7% 0.4% 5 vol 1:1EtOH:H2O 375 mg (75%) 97.9% 1.0% 0.7% 0.3% 5 vol 2:1 EtOH:H2O 354 mg(71%) 97.6% 1.0% 0.6% 0.4% 5 vol 3:1 EtOH:H2O 369 mg (74%) 97.9% 0.9%0.5% 0.3% 5 vol

The 5 vol EtOH/Water slurries were very thick and not easily handled.Since the purity of the solids was comparable from all the trials(slight variations are likely due to the quality of the filtration andwash), the 100% IPA conditions were scaled up as they offered a highrecovery and the resulting suspension was easily handled.

An initial scale up of the preferred slurry gave (92% recovery) withHPLC purity of 96.4% (Impurity levels of 1.2%, 0.7%, 0.4%). Liquorsanalysis showed them to be enriched in all of the main impurities—72%(8.6%, 3.8%, 3.4%) by HPLC. This was deemed a suitable purificationmethod offering a high recovery and the material was use tested in thefollowing stage to ensure tracking and removal of the impurities wasachieved downstream (>99% at stage 3, no single impurity>0.5%).

The slurry proved scalable when remaining crude stage 2 material (70%assay) was water slurried to remove inorganics, and then slurried in IPAto give material of improved purity (97% by NMR assay, 76% yield forstage 2, 96.8% by HPLC, impurities at 1.1%, 0.8%, 0.4%).

GMP Raw Material Synthesis

A large scale, stage 2 reaction was carried out to supply the GMPcampaign. The reaction progressed as expected to provide crude productthat was water slurried and filtered to provide stage 2 that was 93%pure by HPLC. This was further slurried in 8 vol IPA and filtered togive stage 2 product (93.7% by HPLC, 92% assay, 66% active yield). Sincethe purity obtained was lower than that observed during the developmentcampaign, a use test was conducted which confirmed that high puritystage 3 obtained was suitable for onward processing (GMP raw material).

A second batch was carried out under identical conditions to give crudeproduct which after a water slurry was 90% pure by HPLC. This materialwas subsequently water slurried and purified by IPA slurry to give 384 gof stage 2 product (93.0% by HPLC, 91% NMR assay, 60% active yield).

A third batch was carried out to resupply the GMP synthesis. The crudeproduct was successfully purified by a water then IPA slurry to deliverstage 2 (79% yield) with an increased purity when compared to previousbatches (97.3% by HPLC, 96% NMR assay).

Experimental

Stage 1 (1 eq. limiting reagent) was charged to a vessel under N₂followed by THF (1 vol wrt stage 1 charge) and TBME (6 vol wrt stage 1charge). Oxalyl chloride was then added dropwise to the vessel (1.5 eq)allowing the exotherm to initially raise the temperature to 35-40° C.and then applying cooling as required to maintain 35-40° C. Immediatelyfollowing the addition the reaction was heated to 40° C. and stirred for2-6 hours. The reaction was sampled and analysed for completion, thencooled to RT and heptane (8 vol wrt stage 1 charge) added givingprecipitation of further solids. The reaction was stirred for 10 min andthen the solids were allowed to settle. The majority of the solvent wasdecanted from the solid which was then washed twice with heptane (2×6vol wrt stage 1 charge), decanting in a similar manner after each wash.The solids were then sampled and analysed. TBME was charged to thevessel (4 vol wrt stage 1 charge) to give a yellow slurry which wascooled to −20° C. using a dry ice/acetone bath. A 2M solution of Me₂NHin THF (2 eq) was added dropwise to the vessel over ˜15 min maintainingthe temperature at −20° C. to −10° C. The reaction mixture was allowedto warm slowly to RT and stirred overnight. Further Me₂NH can be addedat this point if required. The reaction was sampled and analysed forcompletion. The reaction was filtered, washing with heptane (2×2 vol wrtstage 1 charge) and the isolated solids dried at 60° C. under vacuum.The crude stage 2 was slurried in water (8 vol wrt stage 1 charge) for2-18 hours and then filtered, washing with water (2 vol wrt stage 1charge). The solids were dried at 60° C. under vacuum to obtain crudestage 2 with <2% w/w water (determined by Karl Fischer titration (KF)).The crude stage 2 was slurried in IPA (10 vol) for 2-18 hrs and thenfiltered, washed with IPA (1 vol wrt mass of crude Stage 2) and ovendried under vacuum at 60° C.

The differences from JNP and the benefits can be summarised as follows:

Step 1

i) Firstly, the use of a THF/TBME solvent system in place of diethylether was less volatile and flammable.

ii) Secondly, the addition of oxalyl chloride was conducted at anelevated temperature, heated to 40° C., giving rise to improvedsolubility and preventing entrapment of stage 1 product in theprecipitate. It also provided a high reaction rate, improved levels ofcompletion and shorter reaction times.iii) Thirdly, the Intermediate 2A was isolated to allow for purificationaway from excess oxalyl chloride.iv) Fourthly, heptane was added to help precipitate Intermediate 2A.Step 2v) By ensuring the amine was used in excess much improved purity andyields were obtained, due to minimal water being present, and hencereduced hydrolysis.vi) Finally, the use of water and IPA slurries provided good purity ofthe Stage 2 product.

Example 4

Stage 3

An initial stage 3 trial reaction was carried out using purified stage 2material (>99% by HPLC) and the supplied reaction conditions. The stage2 input material was found to be largely insoluble in THF, and so ratherthan adding a solution of stage 2 to LiAlH₄ the reverse addition wascarried out. 4 eq of LiAlH₄ was used as a 1M solution in THF with theaddition made at 20-25° C., over ˜2 hrs. At this point 10% product wasobserved with several intermediate species present. The reaction washeated at reflux for ˜7 hrs to give 93% product conversion (by HPLC).The reaction was worked up to give crude stage 3 product (˜90% by HPLC,˜90% by NMR, ˜87% corrected yield).

A trial reaction was carried out in which the LiAlH₄ charge employed wassuccessfully reduced (3 eq vs 4 eq). It was hoped this would benefit thework up by reducing the quantity of Li and Al salts generated. Afterprolonged heating at reflux (10-18 hrs) the reaction intermediate waslargely consumed (2-3% remaining) with ˜95% product by HPLC.

Workup Development

Although the first trial reaction was successfully worked up usingRochelles salt, the volumes employed were very high (˜100 vol) and thisprocedure would not form a viable process for scale up. A variety ofalternative workup procedures were examined in order to try and reducethe volumes required and aid removal of Li/Al salts.

A reduced volume quench was trialed with EtOAc and then Rochelles salt.Grey solids were present as a thick paste which settled to the bottom ofthe flask. While filtration failed, the liquors could be decanted andthe solids re-slurried in THF/EtOAc to extract the product. An aqueousworkup was then carried out and the product isolated by concentration.This yielded product of good purity (90-95% by NMR) in good yield (94%uncorrected for purity). However the process was not readily amenable toscale up.

A reaction was quenched by addition of EtOAc and then sat. Na₂SO₄ in thepresence of anhydrous Na₂SO₄ to act as a binding agent. The reactiongave granular solids which could be readily filtered. An aqueous workupwas then carried out and the product isolated by concentration. A goodyield was obtained (˜94% uncorrected for purity) but the productcontained higher levels of the main impurity by NMR (10% vs 2-4% usuallyobserved).

A reaction was quenched with 20% AcOH at 0° C. leading to the formationof a gel which could not be filtered. The reaction was abandoned.

A reaction was quenched with EtOAc and then 20% citric acid to givesolids which could be separated by filtration. The liquors wereconcentrated to obtain the product. While this procedure was slightlylower yielding (˜77% uncorrected for purity), the product was of veryhigh purity (>95%).

A further reaction was quenched by the addition of EtOAc and then water(3 mL per g of LiAlH₄ in THF). A gel formed which could not be readilyfiltered and the reaction was abandoned.

Finally, a reaction was quenched by the Fieser method. An addition ofwater (1 mL per g of LiAlH₄) was made then 15% NaOH (1 mL per g ofLiAlH₄) and finally water (3 mL per g of LiAlH₄). This gave solids whichcould be filtered from the reaction mixture. The liquors were thenpartitioned and concentrated in vacuo (87% yield, 90-95% by NMR).

These Experiments are summarised in Table 15 below:

TABLE 15 Summary of alternative workup conditions Workup # procedureYield Purity 3.1 EtOAc 2.8 g (94% 90-95% by NMR quench uncorrected)RocheIles salt (reduced vol.) 3.2 EtOAc 2.8 g (94% ~80-85% by NMR,quench in uncorrected) 10% imp. presence of Na₂SO₄ 3.3 20% AcOH Emulsion(reaction to waste) quench 3.4 EtOAc i) 529 mg (71% >95% purity by NMRquench uncorrected) 20% citric ii) 2.3 g (77% acid uncorrected) 3.5Water Emulsion (reaction to waste) quench 3.6 Water/NaOH i) 615 mg (83%90-95% by NMR quench uncorrected) ii) 2.6 g (87% uncorrected)

Both quenches with citric acid and NaOH gave solids that could bereadily filtered from the reaction mixture and required minimal solventvolumes. While the conditions with NaOH were higher yielding (˜10%), theproduct obtained from this procedure was less pure and would likelyrequire further purification before use in the next stage. The loweryield with citric acid was likely due to some precipitation of theproduct citrate salt. This had a purifying effect with clean productobtained directly after concentration. These conditions were chosen forscale up and it was hoped that further optimisation of the citric acidcharge would enable clean product to be isolated in high yield from thisprocess.

The reaction was repeated with a slightly reduced citric acid charge inorder to try and maximise the recovery. This reaction yielded product in57% yield with a further 20% yield obtained by re-slurry of the filtercake in THF (both samples 97.7% by HPLC).

The reaction was scaled up. However during the EtOAc quench, where thereaction was previously seen to thicken, the reaction gummed in theflask to form a thick mass which restricted mixing. While the additionof citric acid then led to the usual slurry/gel this did not represent aviable process. This reaction was worked up with the filter cakere-slurried in THF to maximise the recovery giving 76% active yield,95.0% by HPLC.

The reaction was repeated in order to develop a better quench and avoidthe gum formation seen with EtOAc. A portion was quenched by addition ofacetone which led to a readily stirred suspension/emulsion with no signof thickening. The citric acid treatment was then carried out to give afilterable mixture. This quench was successfully carried out on theremainder of the reaction and worked up to provide crude product in goodyield (71% assay, 82% corrected yield, 98.0% by HPLC).

After the quench the reaction mixture was generally found to be pH 8/9.As part of the workup optimisation process different pHs wereinvestigated. A reaction was split for workup with half receiving aslightly reduced citric acid charge (to obtain pH 11/12 after quench)and the other half taken to pH 7 by addition of further citric acid. ThepH 11 reaction was worked up to give material of 85% NMR assay (73%yield) with the pH 7 reaction giving 60% NMR assay (62% yield). It wasclear from this result that obtaining the correct pH after quench wascritical in order to give a >70% yield. By reducing citric acid chargeonly slightly (still approx. 2 vol of 20% citric acid) an approx. 8%increase in yield was obtained. With this information in hand the pH offuture reactions was monitored during the quench in order to ensure themixture remained strongly basic.

Purification Development

A purification screen was carried out using 100 mg portions of crudePsilocin product which were slurried in 10 vol of solvent with heatcycling to 60° C. The slurries were cooled to RT over the weekend andthen any solids collected by filtration. Stability to acid and base wasalso tested with the view to carrying out an acid/base workup. Theresults of the screen are presented in Table 16 below:

TABLE 16 Psilocin purification screen. (Input purity and 3 main impuritylevels: 90.2%, 3.8%, 0.9%, 0%). Recovery HPLC Purity (and main SolventObservations (approx.) impurity levels) MeOH Solution at RT n/a n/a EtOHSolution at 60° C. 35 mg 97.2%, 0.4%, 1.1%, 0.8% Precipitate at RT IPASlurry at 60° C. 51 mg 97.6%, 0.5%, 0.5%, 1.1% MeCN Solution at 60° C.46 mg 96.8%, 0.6%, 0.4%, 1.6% Precipitate at RT EtOAc Slurry at 60° C.58 mg 97.1%, 0.9%, 0.2%, 1.3% ^(i)PrOAc Slurry at 60° C. 58 mg 98.2%,0.8%, 0.2%, 0.4% Toluene Slurry at 60° C. 70 mg 93.3%, 3.6%, 0.2%, 0.8%Heptane Slurry at 60° C. 77 mg 91.3%, 3.8%, 0.2%, 0.7% Acetone Solutionat 60° C. 30 mg 97.7%, 0.4%, 0.3%, 0.9% Precipitate at RT MEK Solutionat 60° C. 24 mg 97.3%, 0.5%, 0.5%, 1.2% Precipitate at RT MIBK Slurry at60° C. 49 mg 97.4%, 0.6%, 0.2%, 1.3% THF Solution at RT n/a n/a TBMESlurry at 60° C. 67 mg 95.5%, 2.0%, 0.1%, 1.5% DCM Solution at RT n/an/a 1M HCl Solution at RT n/a 83.7%, new imp 8% 1M KOH Slurry at RT(black) n/a 89.1%, 5.4%, 0.9%, 2.2%

The first of the three highlighted impurities corresponded with the moststable reaction intermediate that is observed at ˜70%, when the LiAlH₄addition is complete (requiring refluxing to convert to product). Thethird impurity was not present in the input and appeared to be generatedduring the slurry procedure. Of the solvents that remained as a slurry,^(i)PrOAc gave the highest purity. Several re-crystallisations werefound with MeCN having the potential to remove impurities during thecrystallisation and having a recovery which had the potential to improveduring development. Some degradation was observed in both acid and basewith the KOH sample rapidly turning black.

Purification of the crude stage 3 material was scaled up using the twomost promising solvents (MeCN and ^(i)PrOAc). The solvent volumes werereduced to a minimum in order to improve the recovery. The results ofthese trials are presented in Table 17 below.

TABLE 17 Further development of MeCN/^(i)PrOAc purification RecoveryHPLC Purity (and main Solvent Observations (%) impurity levels) MeCN (5vol) Recryst in 5 512 mg 97.6%, 0.7%, 0.8%, 0% vol (51%) ^(i)PrOAc (3vol) Slurry in 3 vol 706 mg 95.8%, 1.3%, 0.6%, 0% (71%)

A re-crystallisation was obtained from MeCN in 5 vol and a hot slurrywas achieved in ^(i)PrOAc at 3 vol (both at 75° C.). The recovery fromMeCN was again poor despite reduced volumes, however the product was ofvery high purity (>>95% by NMR). The recovery from ^(i)PrOAc was betterwith a large increase in product purity when analysed by NMR (˜95%).

Although HPLC and NMR purity of material from the iPrOAc slurry washigh, a low assay value (85% by NMR assay) was observed. In order toimprove the assay value of the material, as well as remove colour, (allmaterials obtained so far were strongly purple, green or brown)purification by silica pad was investigated.

Crude Psilocin (71% assay, 98.0% by HPLC) was passed through 4 eq ofsilica eluting with THF. An 80% recovery of an off white solid withslightly improved HPLC purity (98.4%) and assay value (˜82% assay) wasobtained. This proved to be an effective means of increasing the productassay value and was therefore included as part of the reaction workup.

A series of ^(i)PrOAc/anti-solvent slurries (Table 18) were thenperformed using the silica treated input (100 mg per slurry) to try andimprove the recovery, whist maintaining chemical purity (input purity98.4%).

TABLE 18 Results of ^(i)PrOAc/anti-solvent additions. Solvent systemRecovery HPLC purity ^(i)PrOAc 78% 99.7% 5 vol 1:1 ^(i)PrOAc:Heptane 70%99.6% 5 vol 1:1 ^(i)PrOAc:TBME 83% 99.7% 5 vol 1:1 ^(i)PrOAc:Toluene 84%99.4% 5 vol ^(i)BuOAc 80% 99.6% 5 vol

Since all purity values were comparable, two solvent systems were chosenfor scale up based on the highest recoveries obtained. The two favouredslurries (TBME and Toluene as anti-solvent) were scaled up (1.0 g perslurry) to better assess the recovery.

TABLE 19 Scale up of favoured purification methods Solvent systemRecovery HPLC purity 1:1 ^(i)PrOAc:TBME 79.9% 99.1% 5 vol 1:1^(i)PrOAc:Toluene 79.4% 99.6% 5 vol

Both of these options provided material of >99% HPLC purity at ˜80%recovery and, combined with a silica pad, appear to provide an effectivemeans of purification for the Psilocin product. Further colour wasremoved into the liquors during the slurry giving Psilocin as a whitesolid. All impurities were effectively removed to below 0.5%. The^(i)PrOAc:TBME slurry was chosen for scale up as this used non-toxic ICHclass 3 solvents.

Scale Up

The developed stage 3 conditions were scaled up and the reactionprogressed to give a completion of 94.4% product with 2.9% of thereaction intermediate present by HPLC after overnight at reflux (typicalof the process). After a silica pad Psilocin was obtained in 83% purityby NMR assay, 66% active yield, 97.0% by HPLC. This material was furtherpurified by slurry in iPrOAc/TBME to give material 100% by NMR assay in62% yield and 99.7% by HPLC.

Due to the crude yield from the reaction being lower than expected (66%vs ˜75%) the filter cake and silica pad were reinvestigated in order totry and recover additional material. However, this was unsuccessful.

The lower than expected yield may have been due to decomposition of theproduct during workup, although previous stress tests had indicated thematerial to be stable under the conditions used. To investigate thisfurther the reaction was repeated. The crude product was isolated beforethe silica pad and additional stress test samples taken to confirmdegradation of the product was not occurring during workup.

The reaction progressed as expected to give completion (93.7% product,2.9% intermediate) and was concentrated yielding crude material (77% NMRassay, 66% active yield). The filter cake was re-slurried in THF/MeOHbut no significant Psilocin was isolated. In order to try and displaceany product that was coordinated to the aluminium salts, further citricacid was added to take the pH to 4 (from pH 8) and the cake re-slurriedin THF, but again no significant Psilocin was isolated. Mass balance wasnot obtained from the reaction with the 66% active yield closelymatching what was previously obtained. This batch was purified by silicapad and slurry in ^(i)PrOAc/TBME to give a 62% yield of high puritymaterial (99.8% by HPLC).

Despite the solvent volumes employed being relatively high and a silicapad being required for removal of aluminium and lithium species, theprocess was still well suited to the required scale.

The stage 3 reaction was further scaled up to process. The reactionproceeded as expected to give completion after 18 hours (˜91% product,˜3% reaction intermediate remaining). Workup by silica pad and slurrygave a 57% yield of high purity Psilocin (>99% by HPLC, 99% NMR assay,0.35% w/w water Karl Fischer).

Experimental

Stage 2 (1 eq. limiting reagent) was charged to the vessel followed byTHF (5 vol wrt stage 2 charge). The mixture was cooled to 0° C. and a 1MTHF solution of LiAlH₄ (3 eq) added dropwise over 30-45 min maintainingthe temperature at 0-20° C. Following the addition, the reaction wasstirred for 30 min at 10-20° C. and then heated to reflux and stirredfor ˜16 hrs. The reaction was sampled and analysed for completion,cooled to 0° C. and quenched by dropwise addition of acetone (9.3 eq.)at 0-30° C. followed by a 20% aq citric acid soln (1.9 vol wrt stage 2charge) at 0-30° C. The pH of the addition was monitored to ensure thatit remains at pH>11 and the addition was stopped early if required. Theresulting suspension was stirred for 1 hr and filtered, washing with THF(2 vol wrt stage 2 charge) to remove Li and Al salts. The filter cakewas slurried in THF (12.5 vol wrt stage 2 charge) for ˜1 hr andfiltered, washing with THF (5 vol wrt stage 2 charge) to recover productfrom the Li and Al salts. The combined organics were dried over MgSO₄and filtered. The filtrate was evaporated in vacuo until approximately10 volumes remained (wrt stage 2 charge) and this solution was appliedto a silica pad (3 eq wrt stage 2 charge). The silica pad was elutedwith THF and the product fractions were combined and evaporated todryness in vacuo. The crude stage 3 (Psilocin) was slurried in 1:1iPrOAc:TBME (5 vol wrt mass at step 18) for 2-18 hrs, filtered, washingwith TBME (2.5 vol wrt mass at step 18) and dried in vacuo at 40° C. toisolate pure Psilocin.

The differences from JNP and the benefits can be summarised as follows:

i) Firstly, Applicant, whilst using THF as a solvent, quenched thereaching using acetone. This lead to a suspension/emulsion withoutthickening.

ii) Secondly, Applicant quenched with citric acid maintaining a basicpH, typically about 11. The pH control ensured high yields wereobtained.

iii) Thirdly, following purification by silica pad, to remove residualLi/Al salts, eluting with THF, a iPrOAc:TBME slurry provides a highlypurified product which was then dried.

Example 5

Stage 4

Initially the literature conditions were used to process a 2.58 g samplegiving ˜88% conversion to Intermediate 4A when analysed by HPLC. Theproduct was purified by the addition of aminopropyl silica andfiltration through Celite. The resulting green oil (5.5 g) was slurriedin DCM giving benzyl transfer and precipitation of the zwitterionicstage 4 (4.1 g, 70% yield, ˜95% by NMR).

Step i

Initial development at this stage was focused on finding an alternativeto ^(n)BuLi that was easier to handle and ideally did not introducefurther lithium into the synthesis. An initial screen of alternativeconditions was carried out including the following bases: LrBuO,K^(t)BuO, NaH, NaHMDS, and NaNH₂. All of the reactions gave product withNaHMDS performing as well as ^(n)BuLi. All of the reactions became verythick with gelling observed and overhead stirring was recommended forefficient stirring.

The initial screen suggested NaHMDS would be a suitable alternative to^(n)BuLi (81% conversion to product/Intermediate 4A). These conditionswere scaled up to 1.5 g alongside a reference reaction with ^(n)BuLi.Overhead stirring was used in both cases.

TABLE 20 Comparison of ^(n)BuLi and NaHMDS Timepoint HPLC - ^(n)BuLiHPLC - NaHMDS 1 hr, −30° C. 6.6% St 3, 78% Int 4A, <1% St 3, 78% Int 4A,1% St 4 4% St 4 2 hr, 0° C. 6.5% St 3, 77% Int 4A, <1% St 3, 76% Int 4A,1% St 4 4% St 4 Crude product 4.89 g 4.38 g <1% St 3, 2% Int 4A, <1% St3, 5% Int4A, 60% St 4 68% St 4 Abbreviations used in the table: St 3 =Stage 3, Int 4A = Intermediate 4A, St 4 = Stage 4

The reaction profile obtained in both cases was very similar with theNaHMDS reaction giving consumption of stage 3. Both reactions werefiltered on Celite to remove a white precipitate and concentrated. ByNMR excess benzyl protons were present in both cases (especially in theexample with ^(n)BuLi) and the isolated yield was >100%. The NaHMDSconditions proved successful giving a favourable reaction profile andwere chosen for further scale up. However, workup and purificationdevelopment was required.

Step ii

HPLC data indicated the material isolated from the trials above usingNaHMDS and ^(n)BuLi had rearranged to give zwitterionic stage 4 uponconcentration. Purification of this material away from thebenzylphosphoric acid by-products and other impurities was attempted byslurry in a number of solvents.

TABLE 21 Trial purification of crude stage 4 product Solvent Massrecovered HPLC Purity DCM White solid 84% St 4 EtOH Gum — EtOAc Gum —IPA White solid 88% St 4 Toluene Gum — TBME White solid 62% St 4 MIBKGum — MeCN Gum — Acetone White solid 86% St 4 Abbreviations used intable: St 4 = Stage 4

Filtration Poor

White solids were obtained from several solvents however the solidsobtained from DCM and TBME turned to a pale purple gum when stored overthe weekend. Those obtained from IPA and acetone remained as freeflowing white solids on storage suggesting that the stability of thesesolids was likely to be higher and that they would allow for easierhandling.

The slurries in IPA and acetone were scaled up to 1 g. However, gummingwas noticed immediately on addition of the solvent. The gum was slowlydispersed by vigorous stirring and eventually showed signs ofcrystallisation, with a white slurry forming after an overnight stir.However, this process was not suitable for scale up. Solids wereisolated in good yield with IPA providing the highest purity.

THF was also investigated as this had advantages in that it was also thereaction solvent. However, when this was trialed initial gum formationwas again observed (isolated ˜80% yield, ˜92% by HPLC). In order to tryand avoid the gum formation and give a more controlled crystallisationthe crude stage 4 was first solubilised in a low volume of DMSO (2 vol).THF was then added to this (10 vol) and the solution stirred over theweekend. This slowly gave precipitation of the product which wascollected by filtration and washed with THF to yield stage 4 (86% yield)with 96% HPLC purity (>95% by NMR).

As the THF crystallisation was successful and it was previously notedthat complete conversion to zwitterionic stage 4 occurred duringconcentration of the reaction liquors (THF/EtOAc) at 40° C., it washoped that the changes of solvent could be avoided and the productcrystallised directly by stirring out the reaction mixture at 40° C.

Two 4 g NaHMDS reactions were carried out with both reactions reachingcompletion with ˜80% conversion to Intermediate 4A. One reaction wasdiluted with EtOAc and the other with THF and both were filtered toremove phosphate by-products. In order to further reduce the phosphateimpurity levels a brine wash was carried out and the organics dried andconcentrated to 10 vol. These solutions were stirred overnight at 40° C.to give conversion to, and precipitation of, stage 4 (˜1% stage 3, ˜0.2%Intermediate 4A, ˜82% stage 4). The solids were collected by filtrationgiving 8.03 g (88% yield) from EtOAc/THF and 5.65 g (62% yield) fromTHF. The brown/grey solids obtained from EtOAc/THF were of lower purity(˜90% by HPLC, 78% assay) when compared to the white solids obtainedfrom THF (97% by HPLC, 88% assay). Analysis of the aqueous layer fromthe THF reaction showed product to be present and additional losses wereincurred to the final THF filtrate.

Due to the higher purity obtained from THF, this solvent wasinvestigated further in order to optimise the recovery. The brine washwas omitted due to product losses to the aqueous layer and the reactionmixture was further concentrated after the reaction to minimise lossesduring the final filtration step. This new procedure was trialed on a 75g scale with portions of the reaction mixture concentrated to 8 vol and6 vol. Upon filtration no difference in yield was noted between the twoportions with an overall yield of 140.4 g (90% by NMR assay, 74% activeyield, 90% by HPLC).

Impurity Tracking

Three main impurities were observed in the isolated product, withidentities for two of these species proposed based on MS data.

The debenzylated impurity (typically ˜2-5% by HPLC) was shown to givepsilocybin during the following hydrogenation and could therefore betolerated at a higher level. The main observed impurity in the isolatedstage 4 (typically ˜5-8% by HPLC) was the anhydride impurity. This wastracked through the subsequent hydrogenation and shown to be readilyremoved by re-crystallisation from water as the highly solublepyrophosphorate impurity that results from debenzylation. The other mainobserved impurity (m/z 295.2 observed by LCMS) was found to becontrolled to less than 2% by limiting the reaction temperature (below−50° C.) and was not observed in Psilocybin after hydrogenation.

The impurity profile of the 140 g batch produced above showed 90.0%stage 4, 6.4% anhydride impurity, 0.2% N-debenzylated impurity and 1.2%of the m/z 295.2 impurity.

GMP Synthesis

The first large scale stage 3 batch (544 g input) was completed usingthe established procedure to give 213.5 g (53% yield, 99% by HPLC). Asecond batch (628.2 g input) was also processed successfully to give295.2 g (66% yield, 99% by HPLC).

Some variability in yield at this stage was noted over 3 large scalebatches (57%, 53% and 66%). This is probably a consequence of minordifferences in the workup and quench procedure.

Experimental

Stage 3 was charged to a vessel followed by THF (15 vol wrt stage 3charge) and cooled to ≤−50° C. using a dry ice/acetone bath. 1M NaHMDSsolution in THF (1.13 eq) was charged maintaining a temperature of ≤−45°C., target<−50° C. The reaction was stirred for 30 minutes at −60 to−50° C. Tetrabenzylpyrophosphate (2.26 eq) was charged to the reactionin a single portion followed by additional THF (20 vol) whilemaintaining the reaction temperature <−30° C. The reaction was warmed to0° C., over 1.5-2 hours and sampled for completion. The reaction wasfiltered to remove phosphate salts washing with THF (8 vol). Thefiltrate was concentrated until 6-8 vol remains and stirred overnight at40° C. to convert Intermediate 4A to stage 4 product. The reaction wassampled for completion and then filtered and the solid washed with THF(2 vol). The stage 4 product was dried in a vacuum oven at 40° C.

The differences from JNP and the benefits can be summarised as follows:

Step i

i) Firstly, sodium hexamethyldisilazide was introduced to supportdeprotonation. This proved an effective alternative to Butyl Lithium,which was easier to handle, and did not introduce further lithium intothe reaction.

ii) Secondly, by diluting the reaction with THF, a much higher purityIntermediate 4A was obtained.

iii) Thirdly, by controlling the reaction temperature at below −50° C.,undesirable mz 295.2 observed by LCMS was controlled to levels of lessthan 2%.

Step ii

iv) Fourthly, by monitoring levels of stage 4A impurities, particularlythe N-debenzylated Stage 4 (Table 7) and anhydride Stage 4 (Table 7), apure product can be produced reproducibly.

v) The intermediate stage 4A to stage 4 conversion can be carried out inthe reaction solvent, avoiding the need for time consuming solventswaps.

vi) Finally, the obtained solid is washed with THF and oven dried toobtain stage 4.

Example 6

Stage 5

Catalyst poisoning was noted during development of this stage and acharcolation step can be included in the process when required toprevent incomplete hydrogenation. However, charcolation is not routinelyrequired.

After sparging with hydrogen for 3 hours typical reactions showed highlevels of completion (>90% product, 3-5% SM remaining). A small amountof water was added to aid solubility and after sparging with hydrogenfor a further 1 hour, consumption of stage 4 was achieved.

A successful reaction was worked up by filtration, followed byevaporation to remove methanol, leaving the product as a thicksuspension in water. Ethanol was added and the solid filtered to givePsilocybin in 69% yield. ¹H NMR confirmed the identity of the productbut indicated a minor related impurity was present. LCMS analysisindicated a purity of 95.2% with the major impurity (4.1%) beingidentified as the pyrophosphoric acid impurity. (Table 7) deriving fromthe anhydride impurity at stage 4. It was later shown that this impuritywas effectively purged during the final product re-crystallisation(Stage 6).

A further reaction was then carried out using stage 4 material from thefinalised THF workup which was 88.0% pure by HPLC and contained 7.3%N-debenzylated stage 4 (converted to product), with none of theanhydride impurity. Again completion was noted and the reaction workedup as previously to give Psilocybin in 46% yield. The low yield wasbelieved to result from precipitation of the product during the catalystfiltration step. ¹H NMR confirmed the identity of the product and HPLCindicated a purity of 98.9%.

Further development of the reaction conditions was carried out tooptimise the water volume employed and minimise product losses duringthe filtration step. After the reaction, a solution was obtained byaddition of 10 volumes of water with heating to 40° C. This allowed forremoval of the catalyst by filtration without incurring product losseson the filter.

Some stage 3 was generated by hydrolysis during the reaction and workupwith levels of approx. 1-2.5% appearing to be typical of the process. Areduction in the stage 3 level was demonstrated during the final productre-crystallisation.

Scale Up

The large scale stage 4 batch (non-GMP) was processed as a single batch(148 g active input). Consumption of stage 4 was achieved with 88%product and 0.9% stage 3 resulting from hydrolysis. The anhydrideimpurity (6.4%) was completely converted to the correspondingpyrophosphoric acid impurity (5.2%).

The large scale hydrogenation was filtered and concentrated to yield 109g of crude product after stripping back from ethanol to reduce the watercontent (˜71% by NMR assay, 86% yield).

Experimental

10% Pd/C (˜50% water wet, type 87L, 0.1×stage 4 charge) was charged to avessel under N₂ followed by Methanol (20 vol wrt stage 4) and Stage 4.The N₂ was replaced with H₂ and the reaction was stirred under H₂(atmospheric pressure) for 1-2 hours. The reaction was sampled forcompletion and then water was added (10 vol wrt stage 4) maintaining atemperature of <25° C. The mixture stirred for a further 1-2 hours underH₂ (atmospheric pressure). The reaction was sampled and checked forcompletion.

If the reaction was incomplete, H₂ was recharged and the reactioncontinued for a further 1-12 hr until completion was observed. Thereaction was then placed under N₂ and warmed to 40° C. and held for15-45 minutes. The reaction was filtered through celite to removecatalyst, washing with methanol (13.3 vol wrt stage 4 charge) and water(6.7 vol wrt stage 4 charge). The filtrate was concentrated in vacuo,azeotroping with ethanol to remove water until a solid was obtained. Thedifferences from JNP and the benefits can be summarised as follows:

Primarily, the reaction is monitored for levels of intermediates byHPLC, using relative retention times (RRT) and completion controlledwith intermediates being present at less than 0.2%. The stage 5pyrophosphoric acid impurity is also carefully monitored to confirm thatit can be controlled in the final re-crystallisation.

The final Stage 6 process is as described in Example 1.

Example 7

Testing Methodology and Protocols

To test for purity etc the following methodology/protocols wereemployed. 7.1 NMR

¹H and ¹³C NMR spectra of Psilocybin in D₂O were obtained using 400 MHzspectrometer. Chemical shifts are reported in ppm relative to D₂O in the¹H NMR (□=4.75 ppm) and relative to MeOH (□=49.5 ppm), which was addedas a reference, in the ¹³C NMR the spectrum. Literature values forPsilocybin are reported in JNP. Analysis of Psilocybin by NMR gave datathat was consistent with the structure and consistent with that reportedin the literature with only minor variations in chemical shifts forprotons near the ionisable groups which is expected as the zwitterionicnature of the compound makes the material very sensitive to smallchanges in pH.

The ¹H NMR and ¹³C NMR data are outlined below and the spectra are shownin FIGS. 10 and 11.

¹H NMR Data (400 MHz, D₂O): 2.79 (s, 3H), 3.18 (t, J=7.4 Hz, 2H), 3.31(t, J=7.4 Hz, 2H), 6.97 (d, J=8.0 Hz, 1H), 7.08 (s, 1H), 7.10 (t, J=8.0Hz, 1H), 7.19 (d, 8.2 Hz, 1H).

¹³C NMR Data (400 MHz, D₂O (+ trace MeOH): 22.3 (1×CH₂), 43.4 (2×CH₃),59.6 (1×xCH₂), 108.4 (1×CH), 108.6 (1×C), 109.5 (1×CH), 119.1 (d,³J_(P-H)=6.7 Hz, 1×C), 123.3 (1×CH2), 124.8 (1×CH), 139.3 (1×C), 146.3(d, ²J_(P-H)=6.7 Hz, 1×C)

7.2 FT-IR

Data was collected on a Perkin Elmer Spectrum Two™ Spectrometer withUATR Two accessory. Analysis of Psilocybin (Batch: AR755) by FT-IRspectroscopy gave a spectrum (FIG. 12) that is consistent with theproposed structure. The broad peak at 3244 cm⁻¹ is typical of an aminesalt. The remainder of the peaks are in the fingerprint region andtherefore can't be assigned individually.

7.3. Mass Spectrometry

The mass spectrum of Psilocybin (AR755) was obtained on a Bruker Esquire3000 plus Ion Trap Mass Spectrometer and was concordant with thestructure. The mass spectrum (FIG. 13) showed a main peak at m/z=284.8and 568.1 that corresponded to (M+H)⁺ and (2M+H)⁺ of Psilocybin. Thisimplied the molecular ion has m/z 284 corresponding to the molecularformula of Psilocybin (C₁₂H₁₇N₂O₄P) (FIG. 14).

7.4 Residue on Ignition

The residue on ignition method follows the pharmacopeia method with oneadjustment. Inconsistent results were obtained when the crucible washeated to 500° C. and it is believed this is due to low volatility ofthe phosphate residues that are generated. The temperature was thereforeincreased to 800° C. for Psilocybin and consistent and accurate resultswere obtained.

7.5 HPLC—Assay and Purity Determination

The HPLC method used for assay, chemical purity and quantifying theimpurities of Psilocybin is a gradient HPLC-UV method and the conditionsare outlined in Table 22. External standards are used forquantification. Approximately 1 mg/mL of Psilocybin was dissolved inPurified Water:MeOH (95:5). Sonicate to fully dissolve.

Purity by HPLC is calculated in the basis of area % and is correlatedagainst a known retention time standard.

Assay by HPLC is calculated on an anhydrous basis, based on weight %versus a standard of known purity and composition.

TABLE 22 Typical HPLC Conditions for Identification, Purity and AssayParameter Conditions System Agilent 1100 series liquid chromatograph orequivalent Column XBridge C18, 4.6 × 150 mm; 3.5 μm (Ex; watersPN:186003034) Flow Rate 1.0 ml · min⁻¹ Injection Volume 5 μl DetectionUV @ 267 nm Column Temperature 30° C. Mobile Phase A - PurifiedWater:Methanol:TFA (95:5:0.1) B - Methanol:Purified Water:TFA (95:5:0.1)Gradient Time (mins) % A % B  0 100  0  2 100  0 15  0 100 20  0 100 22100  07.6 Residual Solvent Content by HRGC

The HRGC method for quantifying residual solvents is a headspace methodand is described in Table 23 below:

TABLE 23 Typical Residual Solvent GC Method Parameter Conditions SystemAgilent 6890/7890 HRGC or similar Column DB-624 60 m × 0.32 mm, 1.80 μmfilm thickness (or equivalent) Oven Program 40° C. (hold for 15 min)then ramp (20° C. min⁻¹) to 200° C. (hold 5 min) Headspace ParametersOven Temp 125° C. Loop Temp 140° C. Transfer Line Temp 150° C. SplitRatio 10:1 Injector temperature 200° C. Detector temperature 250° C.,FID Head pressure 15 psi, constant pressure Carrier gas Nitrogen Columnflow 2.0 ml · min⁻¹ @ 40° C. Internal Standard 1,2-Difluorobenzene

Levels of the following solvents and reagents are determined: Methanol,Ethanol, THF and Toluene.

7.7 Melting Point by DSC

DSC data was collected on a PerkinElmer Pyris 6000 DSC (or similar). Theinstrument was verified for energy and temperature calibration usingcertified indium. The sample was weighed (typically 0.5 to 3.0 mg) intoa pin-holed aluminium sample pan. The pan was crimped with an aluminiumpan cover. The pan was heated at 20° C./min from 30 to 300° C. with apurge of dry nitrogen at 20 mL/min. During the melting point procedure,each batch of Psilocybin Polymorph A or A′ exhibited two endothermicevents the latter; the first of which was attributed to solid-solidtransition of Polymorph A or A′ to Polymorph B, and the second of whichwas attributed to melting of Polymorph B.

7.8 Polymorphism by XRPD

The solid state form of Psilocybin is determined by XRPD. XRPDdiffractograms were collected on a diffractometer (such as a PANalyticalX'Pert PRO or equivalent) using Cu Kα radiation (45 kV, 40 mA), θ-θgoniometer, focusing mirror, divergence slit (½″), soller slits at bothincident and divergent beam (4 mm) under ambient conditions. The datacollection range was 3-35 °2θ with a continuous scan speed of 0.2°s⁻¹.The resulting diffractogram is compared to that of a referencediffractogram of Polymorph A or A′ to ensure that it is concordant (FIG.7a or 7 b respectively).

7.9 Thermo-Gravimetric Analysis (TGA)

TGA data was collected on a PerkinElmer Pyris 1 TGA (or similar). Theinstrument was calibrated using a certified weight and certified Alumeland Perkalloy for temperature. A predefined amount of sample (typicallyca. 5 mg) was loaded into an aluminium crucible, and was heated at 20°C./min from ambient temperature to 400° C. A nitrogen purge at 20 mL/minwas maintained over the sample.

7.10 Loss on Drying

Determine in duplicate the loss on drying of the sample using a 1 gportion, accurately weighed, dried at 70° C., under vacuum to constantweight.

Calculation:

${\%\mspace{14mu}{Loss}\mspace{14mu}{on}\mspace{14mu}{Drying}} = {\frac{\left( {W_{INTIAL} - W_{FINAL}} \right)}{W_{SAMPLE}} \times 100}$Where:W_(INITIAL)=Initial weight of dish and sample prior to drying (g)W_(FINAL)=Final weight of dish and dried sample (g)W_(SAMPLE)=Weight of sample (g)

Example 8

Forced Degradation Studies

Psilocybin drug substance was stressed under various conditions insolution and in the solid state to provide samples to evaluateanalytical method selectivity.

The forced degradation study was performed on Psilocybin; based on therequirements of ICH Q1A(R2). Testing under stressed conditions hasprovided information on potential degradation pathways and the intrinsicstability of Psilocybin. The optimised analytical method employeddemonstrated specificity to Psilocybin; it was shown to be suitable andchanges to identity, purity and potency of the product can be detectedusing this method. The method used has also been shown to be free frominterferences from likely impurities and degradation products inaccordance with ICH Q2(R1) (Validation of Analytical Procedures) withreference to specificity. Therefore, the HPLC method is deemed suitablefor determining purity of Psilocybin and related impurities.

The control sample of Psilocybin was stable in solution over the studyperiod (study period was 7 days for non-photostability samples).Psilocybin degraded slowly when heated in solution producing psilocin asthe major impurity. Psilocybin was also stable under acid conditions atroom temperature. However, at 60° C. a slow and steady degradation wasobserved producing psilocin as the main impurity. Psilocybin wasslightly unstable at room temperature in the presence of base with slowdegradation to a range of impurities over the study period. Only verylow levels of impurities were formed under the peroxide conditions withthe overall purity dropping by ˜0.5%. In the solid state, slow chemicaldegradation was noted (3 days at 150° C.) predominantly producingpsilocin (stage 3) as an impurity. Psilocybin was stable underphotostability conditions both as a solid and when in solution.

Stability Studies

Stability studies were undertaken with two batches of Psilocybin asshown in Table 24.

TABLE 24 Intended time Study number/ Drug Substance Site of Lot Storagepoints/Study Study start Lot No. Packaging Manufacture Use ConditionStatus ON- GM764B Double food grade Onyx Ref. 2-8° C. 1, 3, 6 monthsYXSTAB0138 Polythene bags. Scientific Std. ongoing Outer Polythene 25°C./60% RH 1, 3, 6 months container ongoing 40° C./75% RH 1, 3, 6 monthsongoing ON- 170231 Double food grade Onyx Clinical 2-8° C. 1, 3, 6, 9,YXSTAB0139 Polythene bags. Scientific 12, 18, 24, 36 Outer Polythenemonths ongoing container 25° C./60% RH 1, 3, 6, 9, 12, 18, 24, 36 monthsongoing 40° C./75% RH 1, 3, 6 months ongoing

Samples were double bagged in food grade polythene bags and sealed in anouter polythene container and placed on storage at 2-8° C., 25° C./60%RH and 45° C./75% RH, a desiccant bag is included between the innerpolythene bags to prevent moisture uptake. Tests for appearance, watercontent, purity and assay were carried out.

The protocols for the two studies are shown in Table 25 and Table 26.

The one month and three months stability data for batch GM764B aredetailed in Table 27 and Table 28 below. The one, three, six, nine andtwelve month stability data for GMP batch 170231 are provided in Table29, Table 30, Table 31, Table 32 and Table 33 respectively below.

TABLE 25 Onyx Stability Trial Protocol Sheet Product: Psilocybin Onyxtrial number: ONYXSTAB0138 Batch number: GM764B Trial due start date: 10MAR. 2017 Test method: N/A Date of manufacture: 6 FEB. 2017 Additionalinformation: 1200 mg of material required in each container. Packagingcomponents: Double polythene bagged lined contained within 300 ml HDPEcontainer (food grade). Insert a desiccant bag between the two polythenebags. Test parameters Appearance Routine Assay (Anhydrous basis) by¹H-NMR tests Water Content by loss on drying Chemical Purity/Impuritiesby HPLC Months 1 3 6 Spares Total 2° C.-8° C. X X X 2 5 25° C./60% RH XX X 2 5 40° C./75% RH X X X 0 3 Date due off 10 APR. 2017 10 JUN. 201710 SEP. 2017 13

TABLE 26 Onyx Stability Trial Protocol Sheet Product: Psilocybin Onyxtrial number: ONYXSTAB0139 Batch number: 170231 Trial due start date: 31MAR. 2017 Test method: SS/PSILOCYBIN/ Date of manufacture: 27 FEB. 2017Additional information: 2200 mg of material required in each container.Packaging components: Double polythene bagged lined contained within 300ml HDPE container (food grade). Insert a desiccant bag between the twopolythene bags. Test parameters Appearance Routine tests Assay (on a drybasis) by HPLC Water Content by loss on drying ChemicalPurity/Impurities by HPLC 1 3 6 9 12 Timepoint month months monthsmonths months 2-8° C. X X X X X 25° C./60%RH X X X X X 40° C./75%RH X XX Date due off 31 APR. 2017 30 JUN. 2017 30 SEP. 2017 31 DEC. 2017 31MAR. 2018 18 24 36 Timepoint months months months Spares Total 2-8° C. XX X 2 10 25° C./60% RH X X X 2 10 40° C./75% RH 1 4 Date due off 30 SEP.2018 31 MAR. 2019 31 MAR. 2020 24

TABLE 27 One Month Stability Data for Batch GM764B Test SpecificationLimit T = 0 T = 1 month T = 1 month T = 1 month Condition N/A N/A 2°C.-8° C. 25° C./60% RH 40° C./75% RH Appearance For information only. Anoff white solid. An off white solid. An off white solid. An off whitesolid. Free from visible Free from visible Free from visible Free fromvisible contamination contamination contamination contamination Assay by¹H-NMR For information only.   97% w/w   99% w/w   98% w/w   96% w/wWater content by loss For information only. 0.86% w/w 0.35% w/w 0.20%w/w 0.14% w/w on drying Chemical Purity By For information only. 99.24%99.24% 99.22% 99.23% HPLC Impurities by HPLC: For Information only.(Quote all GT 0.05%) RRT 0.86  0.05%  0.05%  0.05%  0.05% RRT 1.46 0.05%  0.09%  0.10%  0.10% RRT 1.59  0.37%  0.35%  0.34%  0.34%(Psilocin) Total Impurities  0.76%  0.76%  0.78%  0.77%

TABLE 28 Three Month Stability Data for Batch GM764B Test SpecificationLimit T = 0 T = 3 month T = 3 month T = 3 month Condition N/A N/A 2°C.-8° C. 25° C.-60% RH 40° C.-75% RH Appearance For information only. Anoff white solid. An off white solid. An off white solid. An off whitesolid. Free from visible Free from visible Free from visible Free fromvisible contamination contamination contamination contamination Assay by¹H-NMR For information only.   97% w/w   97% w/w   99% w/w   97% w/wWater content by loss For information only. 0.86% w/w 0.26% w/w 0.08%w/w 0.14% w/w on drying Chemical Purity By For information only. 99.24%99.31% 99.27% 99.26% HPLC Impurities by HPLC: For Information only.(Quote all GT 0.05%) RRT 0.86  0.05% LT 0.05% LT 0.05% LT 0.05% RRT 1.46 0.05%  0.10%  0.09%  0.10% RRT 1.59  0.37%  0.37%  0.36%  0.37%(Psilocin) Total Impurities  0.76%  0.69%  0.73%  0.74%

TABLE 29 One Month Stability Data for Batch 170231 Test SpecificationLimit T = 0 T = 1 month T = 1 month T = 1 month Condition N/A N/A 2°C.-8° C. 25° C./60% RH 40° C./75% RH Appearance For information only. Anoff white solid. An off white solid. An off white solid. An off whitesolid. Free from visible Free from visible Free from visible Free fromvisible contamination contamination contamination contamination ChemicalPurity By For information only. 99.28% 99.20% 99.16% 99.17% HPLCImpurities by HPLC: For Information only. (Quote all GT 0.05%) RRT 1.49 0.06%  0.05%  0.05%  0.06% RRT 1.62  0.39%  0.36%  0.37%  0.36%(Psilocin) RRT 1.70  0.05% LT 0.05% LT 0.05% LT 0.05% Impurity at RRT1.89 LT 0.05% LT 0.05% LT 0.05% LT 0.05% Impurity at RRT 2.45 LT 0.05%LT 0.05% LT 0.05% LT 0.05% Impurities LT 0.05%  0.22%  0.39%  0.42% 0.41% Total Impurities  0.72%  0.80%  0.84%  0.83% Assay by HPLC Forinformation only 98.65% w/w 98.76% w/w 97.98% w/w 98.52% w/w (on a drybasis) Water content by loss For information only.  0.32% w/w  0.27% w/w 0.17% w/w  0.19% w/w on drying

TABLE 30 Three Month Stability Data for Batch 170231 Test SpecificationLimit T = 0 T = 3 months T = 3 month T = 3 month Condition N/A N/A 2°C.-8° C. 25° C./60% RH 40° C./75% RH Appearance For information only. Anoff white solid. An off white solid. An off white solid. An off whitesolid. Free from visible Free from visible Free from visible Free fromvisible contamination contamination contamination contamination ChemicalPurity By For information only. 99.28% 99.30% 99.31% 99.17% HPLCImpurities by HPLC: For Information only. (Quote all GT 0.05%) RRT 0.69LT 0.05%  0.05% LT 0.05% LT 0.05% RRT 1.49  0.06%  0.05%  0.05%  0.06%RRT 1.62  0.39%  0.37%  0.36%  0.39% (Psilocin) RRT 1.70  0.05% LT 0.05%LT 0.05% LT 0.05% Impurity at RRT 1.89 LT 0.05% LT 0.05% LT 0.05% LT0.05% Impurity at RRT 2.45 LT 0.05% LT 0.05% LT 0.05% LT 0.05%Impurities LT 0.05%  0.22%  0.22%  0.27%  0.34% Total Impurities  0.72% 0.70%  0.69%  0.79% Assay by HPLC For information only 98.65% w/w98.45% w/w 99.46% w/w 98.64% w/w (on a dry basis) Water content by lossFor information only.  0.32% w/w  0.17% w/w  0.01% w/w  0.19% w/w ondrying

TABLE 31 Six Month Stability Data for Batch 170231 Test SpecificationLimit T = 0 T = 6 months T = 6 months T = 6 months Condition N/A N/A 2°C.-8° C. 25° C.-60% RH 40° C.-75% RH Appearance For information only. Anoff white solid. An off white solid. An off white solid. An off whitesolid. Free from visible Free from visible Free from visible Free fromvisible contamination contamination contamination contamination ChemicalPurity By For information only. 99.28% 99.20% 99.19% 99.12% HPLCImpurities by HPLC: For Information only. (Quote all GT 0.05%) RRT 0.69LT 0.05%  0.06%  0.06%  0.06% RRT 1.49  0.06%  0.07%  0.07%  0.08% RRT1.62  0.39% LT 0.05% LT 0.05% LT 0.05% (Psilocin)  0.35%  0.34%  0.38%RRT 1.70  0.05% LT 0.05% LT 0.05% LT 0.05% Impurity at RRT 1.89 LT 0.05%LT 0.05% LT 0.05% LT 0.05% Impurity at RRT 2.45 LT 0.05% LT 0.05% LT0.05% LT 0.05% Impurities LT 0.05%  0.22%  0.32%  0.34%  0.36% TotalImpurities  0.72%  0.80%  0.81%  0.88% Assay by HPLC For informationonly 98.65% w/w 97.97% w/w 98.04% w/w 100.10% w/w (on a dry basis) Watercontent by loss For information only.  0.32% w/w  0.06% w/w  0.32% w/w 2.26% w/w on drying

TABLE 32 Nine Month Stability Data for Batch 170231 Test SpecificationLimit T = 0 T = 9 months T = 9 months Condition N/A N/A 2° C.-8° C. 25°C.-60% RH Appearance For information only. An off white solid. An offwhite solid. An off white solid. Free from visible Free from visibleFree from visible contamination contamination contamination ChemicalPurity By For information only. 99.28% 99.16% 99.16% HPLC Impurities byHPLC: For Information only. (Quote all GT 0.05%) RRT 0.69 LT 0.05% LT0.05% LT 0.05% RRT 1.49  0.06% LT 0.05% LT 0.05% RRT 1.62  0.39%  0.07% 0.05% (Psilocin)  0.06%  0.06% RRT 1.70  0.05%  0.37%  0.37% Impurityat RRT 1.89 LT 0.05% LT 0.05% LT 0.05% Impurity at RRT 2.45 LT 0.05% LT0.05% LT 0.05% Impurities LT 0.05%  0.22% LT 0.05% LT 0.05%  0.34% 0.35% Total Impurities  0.72%  0.84%  0.84% Assay by HPLC Forinformation only 98.65% w/w 97.53% w/w 98.12% w/w (on a dry basis) Watercontent by loss For information only.  0.32% w/w  0.21% w/w  0.10% w/won drying

TABLE 33 Twelve Month Stability Data for Batch 170231 Test SpecificationLimit T = 0 T = 12 months T = 12 months Condition N/A N/A 2° C.-8° C.25° C./60% RH Appearance For information only. An off white solid. Anoff white solid. An off white solid. Free from visible Free from visibleFree from visible contamination contamination contamination ChemicalPurity By For information only. 99.28% 99.25% 99.25% HPLC Impurities byHPLC: For Information only. (Quote all GT 0.05%) RRT 0.69 LT 0.05% LT0.05% LT 0.05% RRT 1.49  0.06% LT 0.05% LT 0.05% RRT 1.62  0.39%  0.37% 0.37% (Psilocin) RRT 1.70  0.05% LT 0.05% LT 0.05% Impurity at RRT 1.89LT 0.05% LT 0.05% ND Impurity at RRT 2.45 LT 0.05% LT 0.05% LT 0.05%Impurities LT 0.05%  0.22%  0.38%  0.38% Total Impurities  0.72%  0.75% 0.75% Assay by HPLC For information only 98.65% w/w 99.63% w/w 98.97%w/w (on a dry basis) Water content by loss For information only.  0.32%w/w  0.49% w/w  0.61% w/w on drying

Over the first 12 months of the ICH stability study Psilocybin hasproven to be chemically stable under low temperature (2-8° C.), ambient(25° C./60% RH) and accelerated (40° C./75% RH) conditions. There hasbeen no change in the appearance and HPLC analysis has also remainedconsistent. The water content has varied in all samples, due to theinitial impact and then aging of the desiccant bags used in the study.

Example 9—Experimental to Form Hydrate A

Psilocybin (200 mg) was charged to a crystallisation tube followed bydeionised water (4 ml). The mixture was equilibrated at 25° C. for 2hours before the solid was isolated by vacuum filtration. The materialwas split into two equal portions. One portion was not subjected tofurther drying to give Hydrate A, lot GK2, by XRPD and DSC(diffractogram and thermogram consistent with FIG. 7d and FIG. 8drespectively).

Example 10—Experimental to Form Polymorph B

Psilocybin Polymorph A (250 mg) was charged to a round bottom flask,heated to 173° C. using an oil bath and held at temperature for 5minutes. The solid was cooled to ambient temperature and isolated togive lot GK3 with a recovery of 93%. Analysis by XRPD and DSC revealedlot GK3 to be Polymorph B (diffractogram and thermogram consistent withFIG. 7c and FIG. 8c respectively).

Example 11—Solid State Investigations

A number of polymorphism investigations were completed. A summary of thesolid forms found is shown in FIG. 17. The majority of the forms foundwere derived from solvent perturbation; in some cases stoichiometricsolvates were isolated and in other cases non-stoichiometric solvates.

Slurries of Polymorph A

Solvent mediated equilibrations of Psilocybin Pattern A were conductedas a primary route into modification of the solid form and to visuallyassess the solubility of the material in a range of 24 solvents between25 and 50° C.

Psilocybin Pattern A (40 mg) was dosed into tubes at room temperatureand solvents as listed in Table 34 were added in aliquots of 0.4 ml (10vol.) to a total volume of 1.2 ml (30 vol.) and observations noted. Themixtures were agitated constantly. Heat cycling was conducted asfollows: 50° C. for 18 hours, cool over 2 hours to 20° C., mature for 4hours, heat to 50° C. for 4 hours, cool to 20° C. over 2 hours, maturefor 18 hours. A repeat 50° C.-20° C. cycle over 24 hours was conductedand the following applied:

Isolation post heating to 50° C. where solids were sufficient=A series

Isolation post cooling to 20° C. where solids were sufficient=B series

All isolated solids were dried in vacuo at 50° C. for 24 hours andanalysed by XRPD. The observations are provided in Table 34.

The API was largely insoluble in the solvents and solvent mixturestested in 30 volumes at 50° C. resulting in heavy suspensions. Water didsolubilise Psilocybin at 50° C.

TABLE 34 Tabulated observations for heat cycling slurry maturations andusing Pattern A blend as input Obs. Obs. Obs. 20° C., 20° C., 20° C.,Obs. XRPD XRPD Entry Solvent 0.4 ml 0.8 ml 1.2 ml 50° C. A series Bseries 1 Cyclohexane Susp. Susp. Susp. Susp. A A 2 Chlorobenzene Susp.Susp. Susp. Susp. A A 3 2-Chlorobutane Susp. Susp. Susp. Susp. A A 4Benzotrifluoride Susp. Susp. Susp. Susp. A A 5 Anisole Susp. Susp. Susp.Susp. A A 6 Nitromethane Susp. Susp. Susp. Susp. C C 7 CPME Susp. Susp.Susp. Susp. A A 8 Heptane Susp. Susp. Susp. Susp. A A 9 TBME Susp. Susp.Susp. Susp. C A 10 MIBK Susp. Susp. Susp. Susp. A A 11 MEK Susp. Susp.Susp. Susp. A A 12 iPrOAc Susp. Susp. Susp. Susp. C C 13 EtOAc Susp.Susp. Susp. Susp. A A 14 Toluene Susp. Susp. Susp. Susp. A A 15 THFSusp. Susp. Susp. Susp. A A 16 CHCl₃ Susp. Susp. Susp. Susp. A A 17 MeOHSusp. Susp. Susp. Susp. D D 18 EtOH Susp. Susp. Susp. Susp. E E 19 IPASusp. Susp. Susp. Susp. F F 20 MeCN Susp. Susp. Susp. Susp. C A 21 WaterSusp. Susp. Susp. Solution n/a A 22 4:1 EtOH/water Susp. Susp. Susp.Susp. A A 23 4:1 THF/water Susp. Susp. Susp. Susp. A Hydrate A 24 4:1IPA/water Susp. Susp. Susp. Susp. A C

Results:

In the figures (FIG. 18 and FIG. 19), “25° C.” denotes isolation of thesolid at 25° C. and “50° C.” denotes isolation of the solid at 50° C.For example, GM832-20_50_A9 represents GM832 entry 20 (MeCN) isolated at50° C.

50° C. Slurries

Entries 1, 2, 3, 4, 5, 7, 8, 10, 11, 13, 14, 15, 16, 22, 23, 24: XRPDdiffractogram broadly consistent with Polymorph A, but with anadditional peak of varying intensity at 18 °2θ.

Entries 6, 9, 12, 20: XRPD diffractogram acquired for the isolatedsolids were broadly consistent (see FIG. 18) with additional peaks at 10°2θ and 13.2 °2θ observed for some samples. This XRPD diffractogram wasdesignated Pattern C. There is no chemotype correlation between thesolvents (CH₃NO₂, TBME, iPrOAc and CH₃CN).

Entry 17: XRPD diffractogram acquired had multiple diffraction peaks(FIG. 19). The XRPD diffractogram was designated Pattern D.

Entry 18: XRPD diffractogram acquired had multiple diffraction peaks(FIG. 19). The XRPD diffractogram was designated Pattern E.

Entry 19: XRPD diffractogram acquired had multiple diffraction peaks(FIG. 19). The XRPD diffractogram was designated Pattern F.

25° C. slurries:

Entries 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 13, 14, 15, 16, 20, 21, 22: XRPDdiffractograms are all similar to that acquired for Polymorph A.

Entries 6, 12, 24: XRPD diffractograms acquired for the isolated solidswere broadly consistent (see FIG. 18) with Pattern C.

Entry 23: XRPD analysis showed Hydrate A had formed.

Entry 17: XRPD diffractogram acquired had multiple diffraction peaks(FIG. 19). The XRPD diffractogram was designated Pattern D.

Entry 18: XRPD diffractogram acquired had multiple diffraction peaks(FIG. 19). The XRPD diffractogram was designated Pattern E.

Entry 19: XRPD diffractogram acquired had multiple diffraction peaks(FIG. 19). The XRPD diffractogram was designated Pattern F.

Analysis of Results:

The XRPD diffractograms for the solids isolated at 25° C. are broadlythe same as for the XRPD diffractograms acquired for the solids isolatedat 50° C.

Patterns D, E and F were derived from alcohols (MeOH, EtOH and IPA).Solvated states were postulated considering an Ethanol Solvate waspreviously isolated during development. The XRPD diffractograms for theEthanol Solvate are not identical, however, given that exact solventlevel variation may deliver varying states of order within the lattice,the comparison between these XRPD diffractograms provides a stronghypothesis that these more significant phase variations are invoked bysolvent entrainment.

XRPD patterns D, E and F (FIG. 19) are all dissimilar to the XRPDdiffractogram for Hydrate (FIG. 7d ).

Direct comparison of the XRPD diffractograms acquired for the MeOH, EtOHand IPA derived solids (Patterns D, E and F—FIG. 19) isolated at the twotemperatures shows conforming diffractograms; the two MeOHdiffractograms are similar while the EtOH and IPA are directlycomparable.

DSC analysis was performed on the isolated solids, and where sufficientsample was available, TGA. The solids that delivered Patterns D, E and Fall features endotherms at ca. 170-180° C. but otherwise proffereddistinct thermal profiles. TGA analysis for the MeOH slurry isolatedsolid showed one protracted mass loss from ca. 25-190° C. (3.1%). Astoichiometric methanol solvate would require 10.3% weight solvent. TGAanalysis of the EtOH slurry isolated solid showed two distinct mass losssteps. The first one occurred before 100° C. (0.3% weight) is consideredto be due to water, and the second larger loss (11.5% weight) due tosolvent. A stoichiometric ethanol solvate requires 13.9% weight solvent.TGA analysis of the IPA slurry isolated solid featured two distinct massloss steps. The first mass loss before 100° C. (0.4% weight) isconsidered to be due to water, while the second larger mass loss (13.9%weight) is considered to be due to residual solvent. A stoichiometricIPA solvate requires 17.5% weight solvent.

Slurries of Amorphous Psilocybin

In order to generate amorphous material a small sample of Psilocybin(0.5 g) was dissolved in water (0.5 L, 1000 vol.), polish filtered andlyophilised. Psilocybin was recovered as an off white fibrous material(lot MC1368A; 412 mg, 82%, XRPD amorphous).

To assess visually solubility of the amorphous API and to induce formmodification, a series of slurry maturations were performed as follows:

Psilocybin (15 mg) was charged to tubes. Solvent was then added atambient temperature (20° C., 0.3 ml, 20 vol.) and the suspensionsagitated. Observations were made. After 1 hour of stirring, samples wereheated to 45° C. for 18 hours and observations made. Samples were heatedto 50° C. for 8 hours and observations were made. The samples wereagitated for 72 hours at 25° C. and subject to a final heat cycle, priorto isolation. Observations are shown in Table 35.

TABLE 35 Observations of amorphous Psilocybin during heat cycling slurrymaturation and form fate Obs. at Obs. at Obs. at Entry Solvent 20° C.45° C. 50° C. XRPD Data A Cyclohexane Susp. Susp. Susp. Semi-crystallineB Chlorobenzene Susp. Susp. Susp. Semi-crystalline C Chlorobutane Susp.Susp. Susp. Pattern B D Benzotrifluoride Susp. Susp. Susp.Semi-crystalline E Anisole Susp. Susp. Susp. Semi-crystalline FNitromethane Susp. Susp. Susp. Pattern B G CPME Susp. Susp. Susp.Semi-crystalline H Heptane Susp. Susp. Susp. Semi-crystalline I TBMESusp Susp. Susp. Semi-crystalline J MIBK Susp. Susp. Susp.Semi-crystalline K MEK Susp. Susp. Susp. Semi-crystalline L iPrOAc Susp.Susp. Susp. Semi-crystalline M EtOAc Susp. Susp. Susp. Semi-crystallineN Toluene Susp. Susp. Susp. Similar to Solvate A O THF Susp. Susp. Susp.Semi-crystalline P Chloroform Susp. Susp. Susp. Similar to Pattern E RMeOH Susp. Susp. Susp. Semi-crystalline S EtOH Susp. Susp. Susp. PatternD T IPA Susp. Susp. Susp. Pattern B U Acetonitrile Susp. Susp. Susp.Amorphous V Water Susp. Susp. Susp. Similar to Pattern A W 4:1EtOH/water Susp. Susp. Susp. Similar to Pattern D X 4:1 THF/water Susp.Susp. Susp. Similar to Pattern A Y 4:1 IPA/water Susp. Susp. Susp.Similar to Pattern A

Results

The majority of solvents returned a solid that was considered to besemi-crystalline (predominantly amorphous with a notable reflection atca. 18 °2θ).

Truly amorphous was returned from equilibration in MeCN.

Polymorph B was returned from chlorobutane, nitromethane and IPA (FIGS.20 and 22).

Pattern D, which was isolated from MeOH in the Polymorph A slurryexperiments discussed above, was returned from the EtOH equilibrationwhereas MeOH in this study returned a semi-crystalline solid.

Solids similar to Pattern A were recovered from water, THF:Water andIPA:Water (4:1).

A solid similar to Pattern D was recovered from EtOH:Water (4:1),supporting the finding of the isolation of Pattern D from EtOH alone.

A solid similar to Pattern E was recovered from Chloroform.

From none of the solvents investigated was true Polymorph A or A′isolated following extended equilibration and thermal maturation ofamorphous Psilocybin.

Example 12—Formulation Development

An initial series of experiments were conducted using formulations asset out in Table 36 below. The objective was to identify suitable singlefiller or combination fillers for large scale formulation.

TABLE 36 Batch No (% w/w) APL-117- APL-117- APL-117- Material Name6085-01 6085-02 6085-03 Psilocybin  1.0  1.0  1.0 Microcrystalline  91.5 49.5  81.5 cellulose Ph 102 Pregelatinised Starch —  45.0 — (Starch1500) Compact Cel MAB — — 10  Hydroxypropyl  3.0  3.0  3.0 Cellulose(Klucel EXF) Sodium Starch  3.0  3.0  3.0 Glycolate Colloidal silicon 0.5  0.5  0.5 Dioxide Magnesium Stearate  1.0  1.0  1.0 (VegetableDerived) Sodium Stearyl — — — Fumarate TOTAL 100.0 100.0 100.0

The outcome, in terms of key physiochemical properties—Material flow,Blend Uniformity, and Content Uniformity are set out in Table 37 below:

TABLE 37 Strength Material flow Blend Content Batch No (mg) (CarrsIndex) Uniformity uniformity APL-117- 1.0 19.1 TOP = 127.9 % Label6085-01 Middle = 106.4 claim = Bottom = 104.5 92.4 Mean = 112.9 AV = 7.9% RSD = 10.9 APL-117- 1.0 19.1 TOP = 115.9 % Label 6085-02 Middle =106.6 claim = Bottom = 106.1 95.2 Mean = 109.6 AV = 5.9 % RSD = 4.9APL-117- 1.0 22.4 TOP = 105.0 % Label 6085-03 Middle = 101.4 claim =Bottom = 98.7 96.3 Mean = 101.7 AV = 4.6 % RSD = 3.8

Whilst batch (APL-117-6085-03) showed good blend uniformity acrossdifferent sample analysed (TOP, MIDDLE and BOTTOM) and very good contentuniformity its flow property (based on Carr's index) were towards thehigh end and it was predicted that the formulation would not accommodatehigher drug loads.

For this reason, a number of alternative formulations were trialed. Theobjective was to consider other filler combinations with the aim ofimproving the powder flow as well as achieving good blend uniformity andcontent uniformity.

Formulations containing less Compactcel MAB and higher amount of glidantcompared to Batch 3 (APL-117-6085-03) were also trialed

These further formulations are set out in Table 38 below.

TABLE 38 Batch No (% w/w) APL-117- APL-117- APL-117- Material Name6085-05 6085-06 6085-07 Psilocybin  1.0  1.0  5.0 Microcrystalline — 89.0  85.0 cellulose Ph 102 Pregelatinised Starch  45.0 — — (Starch1500) Compact Cel MAB —  5.0  5.0 Microcrystaline  49.5 — — CelluloseCEOLUS UF 702 Sodium Starch  3.0  3.0  3.0 Glycolate Colloidal silicon 0.5  1.0  1.0 Dioxide Sodium Stearyl  1.0  1.0  1.0 Fumarate TOTAL100.0 100.0 100.0

The results for these Batches are shown in Table 39 below:

TABLE 39 Strength Material flow Blend Content Batch No (mg) (CarrsIndex) Uniformity uniformity APL-117- 1.0 20.9 TOP = 130.0 % Label6085-05 Middle = 105.4 claim = Bottom = 107.2 88.3 Mean = 114.2 AV =16.5 % RSD = 12.6% APL-117- 1.0 20.0 TOP = 107.0 % Label 6085-06 Middle= 96.2 claim = Bottom = 95.5 96.2 Mean = 99.6 AV = 10.5 % RSD = 6.5APL-117- 5.0 24.3 TOP = 91.5 % Label 6085-07 Middle = 94.2 claim =Bottom = 94.8 96.0 Mean = 93.5 AV = 11.9 % RSD = 7.0

APL-117-6085-05 failed to achieve good blend uniformity, and also failedon content uniformity criteria.

APL-117-6085-06 and APL-117-6085-07 both exhibited improved powder flow,but the blend uniformity for both formulations was poorer thanAPL-117-6085-03.

As a consequence, Applicant looked at modified excipients and moreparticularly silicified fillers with different particle sizes. Theseformulations are set out in Table 40 below:

TABLE 40 Batch No (% w/w) Material Name APL-117-6085-11 APL-117-6085-12Psilocybin  5.0  1.0 Prosolv 50  10.5  15.5 Prosolv 90  80.0  79.0Sodium Starch  3.0  3.0 Glycolate Colloidal silicon  0.5  0.5 DioxideSodium Stearyl  1.0  1.0 Fumarate TOTAL 100.0 100.0

Prosolv is a silicified microcrystalline cellulose, and the two variantswere selected to determine if particle size affected outcome. Comparedto standard microcrystalline cellulose (typical size range, depending onsieving, is 80-350 microns) the Prosolv has a finer particle sizedistribution, and that gives an increased surface area. The increasedsurface area it was hypothesised might provide superior flow andincreased compaction together with improved content uniformity andstability in the formulation. The ratio of Prosolv 50 and Prosolv 90 wasto produce a particle size distribution across both finer and coarserparticles.

The results are set out in Table 41 below

TABLE 41 Strength Material flow Blend Content Batch No (mg) (CarrsIndex) Uniformity uniformity APL-117- 5.0 24.3 TOP = 103.4 % Label6085-11 Middle = 100.4 claim = Bottom = 100.2 94.1 Mean = 101.5 AV = 6.0% RSD = 2.0 APL-117- 1.0 21.1 TOP = 101.9 % Label 6085-12 Middle = 98.4claim = Bottom = 99.9 100.5 Mean = 100.1 AV = 5.8 % RSD = 3.8%

It can be seen that the key parameters of content uniformity (greaterthan 90%, and in fact greater than 94%) and AV (less than 10, and infact less than 7) are excellent as is the consistency in blenduniformity (greater than 95% allowing for error).

The invention claimed is:
 1. A method for manufacturing crystallinePolymorph A of psilocybin from psilocin at a scale greater than 100 g,comprising: (i) reacting psilocin with tetrabenzylpyrophosphate to formbenzyl 3-[2-(benzyldimethylazaniumyl)ethyl]-1H-indol-4-yl phosphate;(ii) reacting the benzyl3-[2-(benzyldimethylazaniumyl)ethyl]-1H-indol-4-yl phosphate withhydrogen in the presence of a catalyst to form psilocybin; and (iii)crystallizing the psilocybin from water; wherein the step (iii) furthercomprises drying the crystallized psilocybin to provide crystallinePolymorph A of psilocybin characterized by X-ray powder diffraction(XRPD) peaks at 11.5±0.1, 12.0±0.1, 14.5±0.1, 17.5±0.1 and 19.7±0.1 °2θ,and wherein the crystallized psilocybin has a chemical purity of greaterthan 97% and no single impurity of greater than 1% as determined by HPLCanalysis.
 2. The method of claim 1, wherein the psilocin is manufacturedby a method comprising: (1) reacting 1H-indol-4-yl acetate with oxalylchloride and dimethylamine to form3-([(dimethylcarbamoyl)carbonyl])-1H-indol-4-yl acetate; and (2)reacting the 3-([(dimethylcarbamoyl)carbonyl)-1H-indol-4-yl acetate withlithium aluminum hydride to form psilocin.
 3. The method of claim 1,wherein step (i) further comprises adding sodium hexamethyldisilazide(NaHMDS).
 4. The method of claim 1, wherein tetrahydrofuran (THF) isused as a solvent in step (i).
 5. The method of claim 1, wherein thestep (i) reaction is initiated at a temperature below −50° C.
 6. Themethod of claim 2, wherein the step (1) reaction comprises: (a) reacting1H-indol-4-yl acetate with oxalyl chloride to form3-(2-chloro-2-oxoacetyl)-1H-indol-4-yl acetate; and (b) reacting the3-(2-chloro-2-oxoacetyl)-1H-indol-4-yl acetate with dimethylamine toform 3-([(dimethylcarbamoyl)carbonyl])-1H-indol-4-yl acetate.
 7. Themethod of claim 6, wherein the step (a) reaction is conducted intert-butyl methyl ether (TBME) and THF.
 8. The method of claim 6,wherein the step (a) reaction is conducted at a temperature of about 30°C.-40° C.
 9. The method of claim 6, wherein the dimethylamine is used inexcess in the step (b) reaction.
 10. The method of claim 9, wherein thestep (b) reaction further comprises separating dimethylamine salts fromthe reaction mixture resulting from reacting the3-(2-chloro-2-oxoacetyl)-1H-indol-4-yl acetate with the dimethylaminethrough a purification step, wherein the purification step comprisesforming a slurry comprising the reaction mixture and filtering thereaction mixture.
 11. The method of claim 10, wherein the purificationstep comprises: slurrying the reaction mixture resulting from step (b)with water, isopropyl alcohol (IPA), or a mixture thereof; filtering theslurry to provide isolated3-([(dimethylcarbamoyl)carbonyl])-1H-indol-4-yl acetate; and drying theisolated 3-([(dimethylcarbamoyl)carbonyl])-1H-indol-4-yl acetate. 12.The method of claim 2, wherein the in step (24) reaction is conducted inTHF.
 13. The method of claim 12, wherein a solution of the lithiumaluminum hydride is added to the3-([(dimethylcarbamoyl)carbonyl])-1H-indol-4-yl acetate.
 14. The methodof claim 12, wherein the step (24) reaction further comprises quenchingwith acetone.
 15. The method of claim 12, wherein the step (24) reactionfurther comprises quenching with citric acid.
 16. The method of claim12, wherein the step (2) reaction further comprises filtering a psilocinsolution through silica and washed washing the silica with in THF. 17.The method of claim 16, wherein the psilocin solution is evaporated todryness, and the resulting solid is slurried in a mixture of isopropylacetate and TBME, filtered, washed with in TBME, and dried to providepsilocin.
 18. The method of claim 6, wherein the reaction mixture fromstep (b) is filtered, washed with heptane, and dried to provide the3-([(dimethylcarbamoyl)carbonyl])-1H-indol-4-yl acetate.
 19. The methodof claim 1, wherein the crystallizing step (iii) comprises combining thepsilocybin with about 10-20 volumes of water, heating with agitation toa temperature of at least 70° C. to provide a solution, polish filteringthe solution, seeding the solution at a temperature of about 70° C.,cooling the solution to a temperature of about 5° C. over a period ofmore than 2 hours, and isolating the crystallized psilocybin.
 20. Themethod of claim 19, wherein the psilocybin is combined with about 10volumes of water.
 21. The method of claim 19, wherein the heating withagitation is at a temperature from 75° C. to 80° C.
 22. The method ofclaim 19, wherein the temperature during the cooling is decreased at arate of about 10° C. an hour.
 23. The method of claim 19, wherein theseeding comprises adding crystalline Hydrate A of psilocybin to thesolution, wherein the crystalline Hydrate A of psilocybin ischaracterized by X-ray powder diffraction (XRPD) peaks at 8.9±0.1,13.8±0.1, 19.4±0.1, 23.1±0.1 and 23.5±0.1 °2θ.
 24. The method of claim19, wherein the isolated crystallized psilocybin is washed several timeswith water.
 25. The method of claim 19, wherein the crystallizedpsilocybin has a chemical purity of greater than 99% as determined byHPLC analysis.
 26. The method of claim 1, wherein the catalyst is apalladium on carbon (Pd/C) catalyst.